EP3830300A1 - Treatment and diagnosis of breast cancer - Google Patents
Treatment and diagnosis of breast cancerInfo
- Publication number
- EP3830300A1 EP3830300A1 EP19773488.2A EP19773488A EP3830300A1 EP 3830300 A1 EP3830300 A1 EP 3830300A1 EP 19773488 A EP19773488 A EP 19773488A EP 3830300 A1 EP3830300 A1 EP 3830300A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cadaverine
- breast cancer
- subject
- ldc
- level
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010006187 Breast cancer Diseases 0.000 title claims abstract description 154
- 208000026310 Breast neoplasm Diseases 0.000 title claims abstract description 154
- 238000011282 treatment Methods 0.000 title claims abstract description 47
- 238000003745 diagnosis Methods 0.000 title abstract description 6
- 108010048581 Lysine decarboxylase Proteins 0.000 claims abstract description 104
- 241000894006 Bacteria Species 0.000 claims abstract description 91
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 64
- 238000000034 method Methods 0.000 claims abstract description 37
- 231100000504 carcinogenesis Toxicity 0.000 claims abstract description 13
- 230000000116 mitigating effect Effects 0.000 claims abstract description 12
- VHRGRCVQAFMJIZ-UHFFFAOYSA-N cadaverine Chemical compound NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 claims description 411
- 206010028980 Neoplasm Diseases 0.000 claims description 89
- 108090000623 proteins and genes Proteins 0.000 claims description 63
- 239000000523 sample Substances 0.000 claims description 61
- 238000012360 testing method Methods 0.000 claims description 41
- 201000011510 cancer Diseases 0.000 claims description 39
- 210000002966 serum Anatomy 0.000 claims description 32
- 241000736262 Microbiota Species 0.000 claims description 30
- 241000588724 Escherichia coli Species 0.000 claims description 29
- 230000003247 decreasing effect Effects 0.000 claims description 24
- 210000003608 fece Anatomy 0.000 claims description 21
- 230000001580 bacterial effect Effects 0.000 claims description 19
- 206010027476 Metastases Diseases 0.000 claims description 17
- 101100398755 Escherichia coli (strain K12) ldcC gene Proteins 0.000 claims description 15
- 230000015572 biosynthetic process Effects 0.000 claims description 15
- 230000008595 infiltration Effects 0.000 claims description 15
- 238000001764 infiltration Methods 0.000 claims description 15
- 201000005389 breast carcinoma in situ Diseases 0.000 claims description 13
- 241000588697 Enterobacter cloacae Species 0.000 claims description 12
- 238000003786 synthesis reaction Methods 0.000 claims description 12
- 241000588729 Hafnia alvei Species 0.000 claims description 10
- 101100276041 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) ctpD gene Proteins 0.000 claims description 9
- 210000000481 breast Anatomy 0.000 claims description 9
- 101150008667 cadA gene Proteins 0.000 claims description 9
- 239000008194 pharmaceutical composition Substances 0.000 claims description 7
- 241000588722 Escherichia Species 0.000 claims description 6
- 241000588914 Enterobacter Species 0.000 claims description 5
- 241000588731 Hafnia Species 0.000 claims description 5
- CJNBYAVZURUTKZ-UHFFFAOYSA-N hafnium(IV) oxide Inorganic materials O=[Hf]=O CJNBYAVZURUTKZ-UHFFFAOYSA-N 0.000 claims description 5
- 230000002265 prevention Effects 0.000 claims description 5
- 239000013074 reference sample Substances 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 137
- 241001529936 Murinae Species 0.000 description 64
- 108020004414 DNA Proteins 0.000 description 57
- 241000282414 Homo sapiens Species 0.000 description 51
- 230000002441 reversible effect Effects 0.000 description 42
- 230000014509 gene expression Effects 0.000 description 37
- 230000002550 fecal effect Effects 0.000 description 29
- 235000018102 proteins Nutrition 0.000 description 25
- 102000004169 proteins and genes Human genes 0.000 description 25
- 241000699670 Mus sp. Species 0.000 description 19
- 239000000047 product Substances 0.000 description 17
- 230000000875 corresponding effect Effects 0.000 description 16
- 210000001035 gastrointestinal tract Anatomy 0.000 description 15
- 210000001519 tissue Anatomy 0.000 description 14
- 238000003556 assay Methods 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 241001465754 Metazoa Species 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 244000005709 gut microbiome Species 0.000 description 12
- 229920000768 polyamine Polymers 0.000 description 12
- 230000004083 survival effect Effects 0.000 description 12
- 230000007705 epithelial mesenchymal transition Effects 0.000 description 11
- 244000005702 human microbiome Species 0.000 description 11
- 244000005700 microbiome Species 0.000 description 11
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 10
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 description 9
- 239000004472 Lysine Substances 0.000 description 9
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 9
- 238000011529 RT qPCR Methods 0.000 description 9
- 230000037396 body weight Effects 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- -1 FgFbpl Proteins 0.000 description 8
- 101000990915 Homo sapiens Stromelysin-1 Proteins 0.000 description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 8
- 102100030416 Stromelysin-1 Human genes 0.000 description 8
- 230000009545 invasion Effects 0.000 description 8
- 235000018977 lysine Nutrition 0.000 description 8
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 description 8
- 238000011160 research Methods 0.000 description 8
- 239000003981 vehicle Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 102100026802 72 kDa type IV collagenase Human genes 0.000 description 7
- 101150082819 ERBB3 gene Proteins 0.000 description 7
- 101000627872 Homo sapiens 72 kDa type IV collagenase Proteins 0.000 description 7
- 102000004369 Insulin-like growth factor-binding protein 4 Human genes 0.000 description 7
- 108090000969 Insulin-like growth factor-binding protein 4 Proteins 0.000 description 7
- 101150009943 Tgfb3 gene Proteins 0.000 description 7
- 229910002092 carbon dioxide Inorganic materials 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 210000004379 membrane Anatomy 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 102000000905 Cadherin Human genes 0.000 description 6
- 108050007957 Cadherin Proteins 0.000 description 6
- 241000282412 Homo Species 0.000 description 6
- 230000007423 decrease Effects 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 210000000981 epithelium Anatomy 0.000 description 6
- 210000002950 fibroblast Anatomy 0.000 description 6
- 210000003205 muscle Anatomy 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 102000007469 Actins Human genes 0.000 description 5
- 108010085238 Actins Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 5
- 241001559542 Hippocampus hippocampus Species 0.000 description 5
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 5
- 229930182816 L-glutamine Natural products 0.000 description 5
- 229930182555 Penicillin Natural products 0.000 description 5
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 5
- 238000000692 Student's t-test Methods 0.000 description 5
- QHNORJFCVHUPNH-UHFFFAOYSA-L To-Pro-3 Chemical compound [I-].[I-].S1C2=CC=CC=C2[N+](C)=C1C=CC=C1C2=CC=CC=C2N(CCC[N+](C)(C)C)C=C1 QHNORJFCVHUPNH-UHFFFAOYSA-L 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 210000004292 cytoskeleton Anatomy 0.000 description 5
- 238000006114 decarboxylation reaction Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 230000011278 mitosis Effects 0.000 description 5
- 229940049954 penicillin Drugs 0.000 description 5
- 230000004962 physiological condition Effects 0.000 description 5
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 229960005322 streptomycin Drugs 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- QOFZZTBWWJNFCA-UHFFFAOYSA-N texas red-X Chemical compound [O-]S(=O)(=O)C1=CC(S(=O)(=O)NCCCCCC(=O)O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 QOFZZTBWWJNFCA-UHFFFAOYSA-N 0.000 description 5
- 210000002700 urine Anatomy 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 239000005700 Putrescine Substances 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 230000006907 apoptotic process Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 230000002354 daily effect Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- IOOMXAQUNPWDLL-UHFFFAOYSA-M lissamine rhodamine anion Chemical compound C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=C(S([O-])(=O)=O)C=C1S([O-])(=O)=O IOOMXAQUNPWDLL-UHFFFAOYSA-M 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000002207 metabolite Substances 0.000 description 4
- 230000009401 metastasis Effects 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 238000001543 one-way ANOVA Methods 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 239000008399 tap water Substances 0.000 description 4
- 235000020679 tap water Nutrition 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 description 3
- 206010001488 Aggression Diseases 0.000 description 3
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 description 3
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 description 3
- 108020000946 Bacterial DNA Proteins 0.000 description 3
- 206010009944 Colon cancer Diseases 0.000 description 3
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 3
- 108091006027 G proteins Proteins 0.000 description 3
- 102000030782 GTP binding Human genes 0.000 description 3
- 108091000058 GTP-Binding Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000007995 HEPES buffer Substances 0.000 description 3
- 206010025323 Lymphomas Diseases 0.000 description 3
- 102000004406 Trace amine-associated receptor 1 Human genes 0.000 description 3
- 108090000946 Trace amine-associated receptor 1 Proteins 0.000 description 3
- 210000003815 abdominal wall Anatomy 0.000 description 3
- 210000000577 adipose tissue Anatomy 0.000 description 3
- 238000011888 autopsy Methods 0.000 description 3
- 239000003613 bile acid Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000000546 chi-square test Methods 0.000 description 3
- 230000007140 dysbiosis Effects 0.000 description 3
- 239000000262 estrogen Substances 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 238000003365 immunocytochemistry Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 108010082117 matrigel Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000007704 transition Effects 0.000 description 3
- 238000010176 18-FDG-positron emission tomography Methods 0.000 description 2
- ZCXUVYAZINUVJD-AHXZWLDOSA-N 2-deoxy-2-((18)F)fluoro-alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H]([18F])[C@@H](O)[C@@H]1O ZCXUVYAZINUVJD-AHXZWLDOSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 102000014156 AMP-Activated Protein Kinases Human genes 0.000 description 2
- 108010011376 AMP-Activated Protein Kinases Proteins 0.000 description 2
- 102000004121 Annexin A5 Human genes 0.000 description 2
- 108090000672 Annexin A5 Proteins 0.000 description 2
- 102100025805 Cadherin-1 Human genes 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 108091007854 Cdh1/Fizzy-related Proteins 0.000 description 2
- 102100037709 Desmocollin-3 Human genes 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 208000027244 Dysbiosis Diseases 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 101000968042 Homo sapiens Desmocollin-2 Proteins 0.000 description 2
- 101000880960 Homo sapiens Desmocollin-3 Proteins 0.000 description 2
- 101001065609 Homo sapiens Lumican Proteins 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 208000008589 Obesity Diseases 0.000 description 2
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 2
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
- 239000012083 RIPA buffer Substances 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 102000011829 Trace amine associated receptor Human genes 0.000 description 2
- 108050002178 Trace amine associated receptor Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 238000003491 array Methods 0.000 description 2
- 208000010572 basal-like breast carcinoma Diseases 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000000035 biogenic effect Effects 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 230000009702 cancer cell proliferation Effects 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000005754 cellular signaling Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 210000001072 colon Anatomy 0.000 description 2
- 230000001332 colony forming effect Effects 0.000 description 2
- 238000004624 confocal microscopy Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001378 electrochemiluminescence detection Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 229940011871 estrogen Drugs 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 102000054090 human LUM Human genes 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 238000002952 image-based readout Methods 0.000 description 2
- 230000000984 immunochemical effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 230000003859 lipid peroxidation Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
- 231100000298 lowest-observed-adverse-effect level Toxicity 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 210000001365 lymphatic vessel Anatomy 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000009826 neoplastic cell growth Effects 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 235000020824 obesity Nutrition 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000036284 oxygen consumption Effects 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 210000004923 pancreatic tissue Anatomy 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 229940121649 protein inhibitor Drugs 0.000 description 2
- 239000012268 protein inhibitor Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 230000002485 urinary effect Effects 0.000 description 2
- JWZZKOKVBUJMES-UHFFFAOYSA-N (+-)-Isoprenaline Chemical compound CC(C)NCC(O)C1=CC=C(O)C(O)=C1 JWZZKOKVBUJMES-UHFFFAOYSA-N 0.000 description 1
- WSYRBHQWMXTCHQ-SFIKJRKMSA-N (2s)-n-[(2r)-1-[[(2s)-1-[[(2r)-1-[[(2r)-1-[[(2s)-1-amino-4-methylsulfanyl-1-oxobutan-2-yl]amino]-3-(1h-indol-3-yl)-1-oxopropan-2-yl]amino]-3-(1h-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1h-indol-3-yl)-1-oxopropan-2-yl]-2-[[( Chemical compound C([C@@H](C(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)[C@@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1NC(=O)CC1)C1=CC=CC=C1 WSYRBHQWMXTCHQ-SFIKJRKMSA-N 0.000 description 1
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
- KJTLQQUUPVSXIM-ZCFIWIBFSA-M (R)-mevalonate Chemical compound OCC[C@](O)(C)CC([O-])=O KJTLQQUUPVSXIM-ZCFIWIBFSA-M 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- HKJKONMZMPUGHJ-UHFFFAOYSA-N 4-amino-5-hydroxy-3-[(4-nitrophenyl)diazenyl]-6-phenyldiazenylnaphthalene-2,7-disulfonic acid Chemical group OS(=O)(=O)C1=CC2=CC(S(O)(=O)=O)=C(N=NC=3C=CC=CC=3)C(O)=C2C(N)=C1N=NC1=CC=C([N+]([O-])=O)C=C1 HKJKONMZMPUGHJ-UHFFFAOYSA-N 0.000 description 1
- PSTVHRSUNBSVIJ-UHFFFAOYSA-N 5-ethyl-5-hexyl-1,3-diazinane-2,4,6-trione Chemical compound CCCCCCC1(CC)C(=O)NC(=O)NC1=O PSTVHRSUNBSVIJ-UHFFFAOYSA-N 0.000 description 1
- RTRQQBHATOEIAF-UHFFFAOYSA-N AICA riboside Natural products NC1=C(C(=O)N)N=CN1C1C(O)C(O)C(CO)O1 RTRQQBHATOEIAF-UHFFFAOYSA-N 0.000 description 1
- QYPPJABKJHAVHS-UHFFFAOYSA-N Agmatine Natural products NCCCCNC(N)=N QYPPJABKJHAVHS-UHFFFAOYSA-N 0.000 description 1
- 102000015404 Amino Acid Receptors Human genes 0.000 description 1
- 108010025177 Amino Acid Receptors Proteins 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 206010003805 Autism Diseases 0.000 description 1
- 208000020706 Autistic disease Diseases 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 108700029181 Bacteria lipase activator Proteins 0.000 description 1
- 206010004446 Benign prostatic hyperplasia Diseases 0.000 description 1
- 244000003027 Bergamotto Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108090000489 Carboxy-Lyases Proteins 0.000 description 1
- 102000004031 Carboxy-Lyases Human genes 0.000 description 1
- 241000905957 Channa melasoma Species 0.000 description 1
- 241000193163 Clostridioides difficile Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 240000007582 Corylus avellana Species 0.000 description 1
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 238000000729 Fisher's exact test Methods 0.000 description 1
- 241000605909 Fusobacterium Species 0.000 description 1
- 206010056740 Genital discharge Diseases 0.000 description 1
- 102000007390 Glycogen Phosphorylase Human genes 0.000 description 1
- 108010046163 Glycogen Phosphorylase Proteins 0.000 description 1
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 description 1
- 101000788239 Homo sapiens Trace amine-associated receptor 2 Proteins 0.000 description 1
- 101000788257 Homo sapiens Trace amine-associated receptor 5 Proteins 0.000 description 1
- 101000674727 Homo sapiens Trace amine-associated receptor 9 Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 208000004882 Intestinal Polyposis Diseases 0.000 description 1
- 208000037396 Intraductal Noninfiltrating Carcinoma Diseases 0.000 description 1
- 235000019766 L-Lysine Nutrition 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- SMEROWZSTRWXGI-UHFFFAOYSA-N Lithocholsaeure Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 SMEROWZSTRWXGI-UHFFFAOYSA-N 0.000 description 1
- 208000004059 Male Breast Neoplasms Diseases 0.000 description 1
- 238000007807 Matrigel invasion assay Methods 0.000 description 1
- 235000010672 Monarda didyma Nutrition 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 108010009711 Phalloidine Proteins 0.000 description 1
- 102100023652 Poly [ADP-ribose] polymerase 2 Human genes 0.000 description 1
- 101710144590 Poly [ADP-ribose] polymerase 2 Proteins 0.000 description 1
- 108091026813 Poly(ADPribose) Proteins 0.000 description 1
- KMNTUASVUKNVJS-UHFFFAOYSA-N Ponceau S (acid form) Chemical compound OC1=C(S(O)(=O)=O)C=C2C=C(S(O)(=O)=O)C=CC2=C1N=NC(C(=C1)S(O)(=O)=O)=CC=C1N=NC1=CC=C(S(O)(=O)=O)C=C1 KMNTUASVUKNVJS-UHFFFAOYSA-N 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000004403 Prostatic Hyperplasia Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 101150060175 Taar4 gene Proteins 0.000 description 1
- 102100039360 Toll-like receptor 4 Human genes 0.000 description 1
- 102100025205 Trace amine-associated receptor 2 Human genes 0.000 description 1
- 102100025204 Trace amine-associated receptor 5 Human genes 0.000 description 1
- 102100021226 Trace amine-associated receptor 9 Human genes 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 1
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 1
- 206010052568 Urticaria chronic Diseases 0.000 description 1
- 230000006682 Warburg effect Effects 0.000 description 1
- 241000193450 [Clostridium] symbiosum Species 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- RTRQQBHATOEIAF-UUOKFMHZSA-N acadesine Chemical compound NC1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 RTRQQBHATOEIAF-UUOKFMHZSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001467 acupuncture Methods 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 230000006536 aerobic glycolysis Effects 0.000 description 1
- QYPPJABKJHAVHS-UHFFFAOYSA-P agmatinium(2+) Chemical compound NC(=[NH2+])NCCCC[NH3+] QYPPJABKJHAVHS-UHFFFAOYSA-P 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 238000011394 anticancer treatment Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002975 chemoattractant Substances 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000024376 chronic urticaria Diseases 0.000 description 1
- 230000004736 colon carcinogenesis Effects 0.000 description 1
- 238000002052 colonoscopy Methods 0.000 description 1
- 238000010293 colony formation assay Methods 0.000 description 1
- 201000002758 colorectal adenoma Diseases 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000001085 cytostatic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000013872 defecation Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000012137 double-staining Methods 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 208000028715 ductal breast carcinoma in situ Diseases 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 201000007280 estrogen-receptor negative breast cancer Diseases 0.000 description 1
- 201000007281 estrogen-receptor positive breast cancer Diseases 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 230000006539 extracellular acidification Effects 0.000 description 1
- 201000010255 female reproductive organ cancer Diseases 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 210000005095 gastrointestinal system Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000006692 glycolytic flux Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 238000010842 high-capacity cDNA reverse transcription kit Methods 0.000 description 1
- 238000010562 histological examination Methods 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000002650 immunosuppressive therapy Methods 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 208000030776 invasive breast carcinoma Diseases 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 229940039009 isoproterenol Drugs 0.000 description 1
- OYIKARCXOQLFHF-UHFFFAOYSA-N isoxaflutole Chemical compound CS(=O)(=O)C1=CC(C(F)(F)F)=CC=C1C(=O)C1=C(C2CC2)ON=C1 OYIKARCXOQLFHF-UHFFFAOYSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 101150041134 ldcC gene Proteins 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000004132 lipogenesis Effects 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- SMEROWZSTRWXGI-HVATVPOCSA-N lithocholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 SMEROWZSTRWXGI-HVATVPOCSA-N 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000010841 mRNA extraction Methods 0.000 description 1
- 201000003175 male breast cancer Diseases 0.000 description 1
- 208000010907 male breast carcinoma Diseases 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 230000004066 metabolic change Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000006677 mitochondrial metabolism Effects 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- CQJSAKJMCVSEGU-UHFFFAOYSA-N n,n-dimethyl-3-pyrido[3,2-b][1,4]benzothiazin-10-ylpropan-1-amine;hydron;chloride Chemical compound Cl.C1=CN=C2N(CCCN(C)C)C3=CC=CC=C3SC2=C1 CQJSAKJMCVSEGU-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000002445 nipple Anatomy 0.000 description 1
- 210000000019 nipple aspirate fluid Anatomy 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 239000000101 novel biomarker Substances 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 231100000496 observed-adverse-effect level Toxicity 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 210000003681 parotid gland Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000035778 pathophysiological process Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000010149 post-hoc-test Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 229960000957 prothipendyl Drugs 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 210000001626 skin fibroblast Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000338 sulforhodamine B assay Toxicity 0.000 description 1
- 238000003210 sulforhodamine B staining Methods 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- IMNIMPAHZVJRPE-UHFFFAOYSA-N triethylenediamine Chemical compound C1CN2CCN1CC2 IMNIMPAHZVJRPE-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 230000002100 tumorsuppressive effect Effects 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
Definitions
- the invention relates to the field of diagnosis and treatment of early stage breast cancer. Specifically, a method is provided for determining whether a subject has early stage breast cancer by measuring the abundance of a bacterium species comprising a DNA sequence coding for a lysine decarboxylase. A method for mitigating breast cancer initiation and/or promotion and/or progression in a subject is also provided.
- Dysbiosis is often reflected as a loss of diversity of the microbiota (e.g.
- Cadaverine (CAD; pentane- 1,5 -diamine) is produced by the decarboxylation of lysine that is performed by lysine decarboxylase (LDC) enzymes.
- LDC lysine decarboxylase
- Human cells code and express LDC and numerous bacterial species of the human microbiome also express LDC either in a constant (LdcC in the LDC operon) or in an inducible (CadA in the Cad operon) fashion [32, 33].
- Bacteria use diamines, like cadaverine or putrescine, generated by the decarboxylation of lysine or arginine, to buffer the pH of their environment [28]. The effects of cadaverine on cancer cells and its role in carcinogenesis are not yet characterized in detail.
- WO2013045826A1 describes that a mixture depleted of cadaverine, putrescine, spermidine and spermine and on the other hand containing agmatine at a concentration higher than that supplied by an average food ration is beneficial in the treatment of pathologies associated with cellular hyperproliferation.
- the invention prvides a method for determining whether a subject has early stage breast cancer or has an in creased probability of having early stage breast cancer by measuring, in a sample derived from said subject, the level of bacterial cadaverine synthesis.
- the invention provides a method for determining whether a subject has early stage breast cancer or has an increased probability of having early stage breast cancer by measuring the abundance of a bacterium species comprising a DNA sequence coding for a lysine decarboxylase (LDC), the level of a DNA sequence coding for an LDC of a bacterium species, or the level of a gene product of a DNA sequence coding for an LDC produced by a bacterium species, in a sample derived from the subject, wherein the sample comprises microbiota from the subject and wherein the bacterium species is part of the microbiota of the subject.
- LDC lysine decarboxylase
- the invention also provides a method for determining whether a subject is at an increased risk of developing breast cancer, by measuring the abundance of a bacterium species comprising a DNA sequence coding for a lysine decarboxylase (LDC), the level of a DNA sequence coding for an LDC of a bacterium species, or the level of a gene product of a DNA sequence coding for an LDC produced by a bacterium species, in a sample derived from the subject, wherein the sample comprises microbiota from the subject and wherein the bacterium species is part of the microbiota of the subject.
- the invention also provides cadaverine for use in the treatment or prevention of breast cancer in a subject and cadaverine for use in mitigating cancer initiation and/or promo tion and/or progression in a subject. The invention is further detailed in the following paragraphs.
- ii) has an increased probability of having early stage breast cancer
- the level of bacterial cadaverine synthesis is assessed by measuring
- compared to the corresponding reference value indicates that the subject has early stage breast cancer or an increased probability of having early stage breast or is at an increased risk of developing breast cancer.
- the breast cancer is stage 0 or 1 breast cancer according to the American Joint Committee on Cancer (AJCC) TNM system.
- the cancer is stage 0 breast cancer and the test sample is compared to a reference value typical of the absence of stage 0 breast cancer.
- the cancer is breast cancer stage 1 the test sample is compared to a reference value typical of the absence of stage 1 breast cancer.
- the DNA sequence coding for LDC is ldcC or cadA.
- test sample is a feces sample.
- the bacterium species is selected from Escherichia, Enterobacter and Hafnia. More preferably the bacterium species is selected from Escherichia cob, Enterobacter cloacae and Hafnia alvei.
- stage 0 breast cancer indicates that the subject has stage 0 breast cancer or an increased probability of having stage 0 breast cancer or is at an increased risk of developing breast cancer.
- stage 0 breast cancer indicates that the subject has stage 0 breast cancer or an increased probability of having stage 0 breast cancer or is at an increased risk of developing breast cancer.
- the gene product is LDC protein.
- the subject is a human, preferably a woman, more preferably a postmenopausal woman.
- cadaverine for use in the treatment or prevention of breast cancer in a sub ject is provided.
- cadaverine for use in mitigating cancer initiation and/or promotion and/or progression in a subject is provided.
- the subject has an increased probability of having early stage breast cancer or is at an increased risk of developing breast cancer. More preferably the subject has early stage breast cancer.
- the level of a DNA sequence coding for an LDC of a bacterium species, or the level of a gene product of a DNA sequence coding for an LDC produced by a bacterium species, is lower in a test sample comprising microbiota from said subject than the corresponding reference value or the level of cadaverine in a test sample from the subject is lower than the corresponding reference value.
- the test sample is feces.
- the bacterium species is selected from Escherichia, Enterobacter and Hafiiia. More preferably the bacterium species is selected from Escherichia coli, Enterobacter cloacae and Hafiiia alvei.
- the DNA sequence coding for an LDC is cadA or ldcC.
- the gene product is LDC protein.
- the invention provides a method for mitigating breast cancer initiation and/or promotion and/or progression in a subject, comprising
- cadaverine administered to the subject to provide or restore physiological serum concentration of cadaverine if
- the subject has early stage breast cancer.
- the test sample is feces.
- the bacterium species is selected from Escherichia, Enterobacter and Hafnia. More preferably the bacte rium species is selected from Escherichia coli, Enterobacter cloacae and Hafnia alvei.
- the DNA sequence coding for an LDC is cadA or ldcC.
- the gene product is LDC protein.
- a pharmaceutical composition comprising cadaverine and at least one pharmaceutically ac ceptable excipient or carrier.
- the pharmaceutical composition is for use in mitigating breast cancer initiation and/or promotion and/or progression.
- the pharmaceutical composition is administered to provide or restore physiological serum concentration of cadaverine in a subject having early stage breast cancer or a lower serum cadaverine level compared to the physiological serum cadaverine.
- Cadaverine treatment reduces breast cancer aggressiveness in vivo.
- CAD cadaverine
- VEH vehicle
- VEH and CAD-treated mice (A) the number and (B) mass of primary tumors were counted and the (C) number and (D) mass of metastases were measured upon autopsy. (E) Upon autopsy, the infiltration rate of the primary tumor was scored.
- F-I Primary tumors were formalin-fixed and were embedded into paraffin, then sections were hematoxylin-eosin stained and were scored for (F) mitosis, (G) mitosis/hpf, (H) nuclear pleomorphism.
- FIG. 1 Cadaverine reduces the proliferation and colony forming ability of breast cancer cells.
- FIG. 3 Cadaverine treatment reverses EMT (epithelial-mesenchymal transition) of breast cancer cells.
- A- B In control and cadaverine-treated 4T1 cells
- FIG. 4 Cadaverine treatment attenuates movement, invasion ability, mitochondrial oxidation and sternness of 4T1 cells.
- B 4T1 cells were treat ed with cadaverine in the concentration indicated for 48 hours and subsequently invasion capacity of the cells was measured using the Corning matrigel invasion chamber. Cells were counted using the Opera HCS system and invasion index was calculated.
- OCR Oxygen consumption rate
- ECAR extra cellular acidification rate
- FIG. 5 TAARs are seemed to be responsible for the effect of cadaverine.
- A Patient data was accessed at kmplot.com. Kaplan-Meier plots show the correlation between the mRNA expression of human TAARs and survival in breast cancer.
- FIG. 6 Cadaverine biosynthesis is suppressed in early stages of breast cancer.
- A Fluman fecal DNA samples were collected from 48 patients with different stages of breast cancer, and from 48 healthy patients. The abundance of DNA coding for CadA and LdC of the indicated bacterial species were determined in the fecal DNA samples by RT-qPCR. Median values are indicated by a line.
- FIG. 7 Cadaverine reverts EMT in breast cancer cell lines.
- Bacterial cadaverine biosynthesis has been found to be suppressed in breast cancer. Bacteria of the human microbiota capable of producing cadaverine may be used as an early indicator of the presence or risk of breast cancer.“Bacterial cadaverine synthesis” refers to the potential of the microbiota to produce cadaverine, prefera bly by the enzymatic decarboxylation of lysine. The level of“bacterial cadaverine synthesis” may depend on the number of bacteria capable of producing cadaverine, e.g the number of bacteria comprising a coding sequence for a lysine decarboxylase enzyme or the rate of expression of the enzyme or the level of activity of the enzyme or the level of the enzyme.
- Bacteria of the gut capable of producing cadaverine are less abundant in the feces of patients with early stage breast cancer.
- the concentrations or amounts of LDC DNA, mRNA or protein are lower in the feces sample of an individual with stage 0 or 1 breast cancer than in a control feces sample.
- The“stage” of a cancer in this description is to be understood as a stage determined using the American Joint Committee on Cancer (AJCC) TNM system staging.
- AJCC American Joint Committee on Cancer
- TNM system American Joint Committee on Cancer staging system was used according to the 7th edition.
- the term“early stage” refers to stage 0 and stage 1, preferably to stage 0 or preferably to stage 1.
- the invention provides a method for diagnosing breast cancer by measuring the abundance of at least one bacterium species which is capable of producing cadaverine under physiological conditions in the human body, in a test sample derived from the subject, wherein the test sample comprises microbiota of said subject.
- the term“abundance” or“level” in general is meant as a proportion of a given specimen in a given pool relative to certain similar specimens.
- the term“abundance” or “level” may refer to a concentration or quantity of the specimen.
- The“abundance” of a bacterium species may refer to the concentration or the number of cells measured in a sample (e.g. a fecal sample).
- the term“abun dance of the DNA sequence coding for lysine decarboxylase (LDC) relates to the proportion of the special seg ment of bacterial DNA making up the DNA sequence coding for lysine decarboxylase in a DNA pool or isolate (e.g. total DNA in a sample or a pool of samples).
- the term“abundance (or level) of the DNA sequence coding for lysine decarboxylase” may relate to the amount of the special segment of bacterial DNA making up the DNA sequence coding for lysine decarboxylase in a sample (test sample or reference sample).
- the abundance or level of an RNA or protein molecule is defined accordingly, i.e. it relates to the amount of the RNA or protein in the sample.
- “abundance” may refer to relative abundance, e.g. the abundance of E. coli in a sample of a patient may be calculated based on the abundance of E. coli in a reference sample.
- Cadaverine is produced by direct decarboxylation of L-lysine catalyzed by lysine decarboxylase.
- a bacterium is capable of producing cadaverine if the genome of said bacterium comprises a DNA sequence coding for a lysine decarboxylase.
- a bacterium is capable of producing cadaverine if its genome comprises a cad and/or ldc operon, more particularly cadA and/or ldcC.
- a bacte rium is capable of producing cadaverine if it expresses a lysine decarboxylase.
- a bacterium that is capable of producing cadaverine produces cadaverine by the decarboxylation of lysine which is catalyzed by LCD.
- a bacterium that is capable of producing cadaverine under physiological condi tions in the human body is capable of producing cadaverine in the gastrointestinal system.
- the bacterium whose abundance is to be measured is a bacterium that is part of the healthy human microbiota, preferably the gut microbiota, preferably the fecal microbiota, more preferably the gut and the fecal microbiota.
- the DNA sequence coding for a lysine decarboxylase may be a constitutive (e.g. ldcC) or an inducible gene (e.g. cadA) and the expression of the protein may be constitutive or inducible.
- the capability of a bacterial spe cies/strain/genus to convert lysine to cadaverine under physiological conditions may be tested in vitro. In an appropriate in vitro assay bacterial lysates and lysine are mixed and at the end of the assay cadaverine is detect ed by e.g. mass spectrometry and the lysine/cadaverine ratio is calculated. Lysine carboxylase activity may be measured as described in Inoue et al.
- A“decreased level of bacterial cadaverine synthesis” in a test sample refers to a decrease of bacterial cadaverine synthesis as compared to a reference level.
- the (corresponding) reference value of abun dance or quantity or level or concentration may be derived from a plurality of samples from individuals not having early stage breast cancer, preferably not having stage 0 or stage 1 breast cancer, preferably not having cancer and most preferably from healthy individuals.
- the corresponding reference value of the serum cadaverine concentration may be the physiological serum concentration of cadaverine.
- the corresponding reference value is the value derived from measurements of the level of the DNA sequence coding for the (same) LDC of the (same) bacterium species in the same type of samples from individuals not having early stage breast cancer, preferably from healthy individuals. Accordingly, a level or concentration of an LDC protein in a test sample is compared to the characteristic value derived from measurements of the level or concentration of the LDC protein in the same type of samples from individuals not having early stage breast cancer, preferably from healthy individuals.
- HPMC Human Pan-Microbe Com munities
- NIH Human Microbiome Project the NIH Human Microbiome Project
- Integrated reference catalog of the human gut microbiome are well-known to the skilled person, and may be found in e.g. Turnbaugh et al. [71] The hu man microbiome project: exploring the microbial part of our in a changing world. Nature 2007; 449(7164): 804-810, e.g the KEGG database.
- human microbiota refers to the microbes capable of living in or on the human body.
- the terms “(human)”“gut microbiota” and“gut microbiome” refer to species of the (human) microbiota living in the (hu man) gastrointestinal tract and the term“fecal microbiota” refers to microbes found in the feces.
- the term “microbiome” and“microbiota” as used in the description may refer to both the (human) microbiota and (hu man) microbiome and the gut/fecal microbiota and gut/fecal microbiome, preferably to the gut/fecal microbiota and microbiome.
- Bacteria capable of producing cadaverine may reside in the gut, on the skin or in other tissues, such as breast tissue and in the feces.
- the abundance of the DNA coding for LdcC and CadA was assessed in human fecal DNA from the experi mental cohort described in the Examples.
- the abundance of Escherichia coli CadA and also E. coli, Enterobacter cloacae and Hafnia alvelii LdcC DNA in breast cancer patients was decreased compared to healthy individuals (Fig. 6A).
- Decreased cadA and ldcC abundance was more pronounced in clinical stage 0 patients as compared to the pool of all patients (Fig. 6A).
- E. coli LdcC protein levels were markedly lower than the levels in the feces of healthy subjects (Fig. 6B).
- the GEO database has been assessed to study LDC expression in human breast cancer. There was no differ ence in LDC mRNA expression between control and breast cancer cases [39-42] or in LDC expression of the normal breast epithelium and cancer epithelium in patients [42, 43]. Rather as an exception, LDC expression was lower in basal-like breast cancer as compared to control (normal) breast epithelium of non-diseased indi viduals [43, 44].
- the kmplot.com database was used to assess how expression of LDC in humans affects the outcome of breast cancer. Differences in LDC expression did affect overall survival of the patients, in grade 1 patients high er expression of LDC was associated with significantly longer survival than lower expression of LDC (Fig. 6C). Interestingly, while LDC expression did not affect survival in ER- PR- patients, higher LDC expression corre lated with better survival in ER+ PR+ patients (Fig. 6D). High expression of human LDC prolongs survival in early stage breast cancer patients, supporting the potential anti-cancer properties of cadaverine.
- cadaverine administered in a dose corresponding to the physiological serum con centration in humans exerted antitumor effects in mice grafted with either human -derived or mouse-derived breast cancer cells.
- the physiological concentration in humans refers to a reference range measured in healthy (i.e. not having breast cancer) individuals and is considered to be 100-800 nM/1 (in the serum) [34, 35].
- the invention provides cadaverine for use in the treatment or prevention of breast cancer in a subject, cadaverine for use in mitigating cancer initiation and/or promotion and/or progression in a subject and cadaverine for use in the treatment or prevention of breast cancer in a subject or in mitigating cancer initiation and/or promotion and/or progression in a subject, wherein in said use physiological serum concentration of cadaverine is provided or restored in said subject.
- physiological serum concentration refers to the serum concentration (or a range of concentrations) measured in healthy subjects.
- Cadaverine plays a tumor suppressor role in breast cancer, in concentrations corresponding to the human reference range.
- cadaverine in the concentrations corresponding to its reference con centration, did not have a cytostatic effect on primary fibroblasts, suggesting that these effects are specific for tumor cells.
- Cadaverine exerts its effects through inhibiting EMT, cellular movement, chemotaxis and metasta sis. It has been also found that cadaverine treatment reduces the aggressivity of breast cancer, as indicated by the decreased rate of mitosis and heterogeneity of nuclear morphology, reduced number of metastases, lower mass and infiltration of the primary tumor. In human patients, rate of mitosis and heterogeneity of nuclear mor phology are part of the Nottingham grading system. Our results suggest that the effect of cadaverine treatment may be detected by the repeated grading of the tumor. Change in nuclear score due to cadaverine treatment is shown on Fig. 1H.
- Infiltration rate of the primary tumors has been found to be lower after cadaverine treatment (Fig. IE).
- “low infiltration” class means that the primeary tumor remained in the mammary fat pads without any attachment to muscle tissues.
- the tumor mass attached to the muscle tissue but did not penetrate to the abdominal wall it was classified as a“medium infiltration” tumor. If the tumor grew into the muscle tissue and totally penetrated the abdominal wall, it was scored as a“high infiltration” tumor.
- histopathological evaluation may be used to determine infiltration rate, e.g. sample specimen after surgery or biopsy may be used.
- Breast cancer is considered systemic when the tumor has infiltrated blood or lymphatic vessels.
- FDG-PET is also suitable to calculate the number of metastases.
- the dose of cadaverine used in the experiments with mice corresponds to the physiological concentration in the serum of healthy humans.
- the human dose of cadaverine to be used may be calculated by known standards, e.g. the Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Thera peutics in Adult Healthy Volunteers from the U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER).
- Cadaverin may be administered in a dose of about 50 nmol/kg bodyweight to 500 miho ⁇ /kg bodyweight, preferably about 50 nmol/kg bodyweight to 250 pmol/kg bodyweight, preferably about 50 nmol/kg bodyweight to 100 pmol/kg bodyweight, preferably about 100 nmol/kg bodyweight to 800 nmol/kg bodyweight, or preferably in a dose providing a serum concentration of 50-2000 nM/1 preferably 100-800 nM/1, more preferably 100-500 nM/1 or 50-500 nM/1.
- cadaverine is administered in a dose restoring physiological serum concentration of cadaverine.
- physiological serum concentration of cadaverine refers to a reference serum concen tration measured in healthy subjects. Loser et al found the serum concentration of cadaverine to be 0.32 ( ⁇ SEM:0.07) nmol/ml in healthy volunteers.
- physiological serum concentration of cadaverine refers to a serum concentration of from about 0.15 nmol/ml to about 0.6 nmol/ml.
- the“physiological serum concentration of cadaverine” refers to a serum concentration of from about 20 nmol/ml to about 0.5 nmol/ml from about 25 nmol/ml to about 0.4 nmol/ml.
- biogenic amines putrescine and cadaverine show in vitro cytotoxicity at concentrations that can be found in foods www.nature.com/scientific reports (2019) 9: 120) non-observed adverse effect level (NOAEL) for cadaverine was found to be 2.5 mM (equivalent to 255.45 mg/kg), while the lowest observed adverse effect level (LOAEL) found to be 5 mM (equivalent to 510.89 mg/kg).
- the IC 50 value for cadaverine was 40.72 ⁇ 1.98 mM. The skilled person may calculate a safe dose for cadaverine from these data.
- Cadaverine may be administered in the form of a pharmaceutical composition, wherein the pharmaceutical composition comprises cadaverine and at least one pharmaceutically acceptable excipient or carrier.
- the pharmaceutical composition comprises cadaverine and at least one pharmaceutically acceptable excipient or carrier.
- Cadaverine is a liquid on room temperature, which fumes and attracts CO2 from the air.
- Cadaverine is soluble in water and alcohol, however, incompatible with acid chlorides, acids, acid anhydrides, strong oxidizing agents, carbon dioxide. These properties must be considered when manufacturing an appropriate dosage form.
- the term“miti gating” refers to delaying, inhibiting, decreasing any of the processes leading to and playing a role in cancer development, including the processes in which cells change to grow and divide continously, spread and invade other tissues.
- primary tumor is used in the generally accepted meaning thereof, indicating the original, or first, tumor(s) in the body. Cancer cells from a primary tumor may spread to other parts of the body and form new, or secondary, tumors.
- Cadaverine teatment (500 nmol/kg) decreased the invasivity of the primary tumors in mice homotopically grafted with 4T1 breast cancer cells (Fig. IE). Histological examination of the primary tumors revealed that cadaverine teatment decreased the rate of mitosis (Fig. 1F-G), the heterogeneity of nuclear morphology (Fig. 1H) and the pathological grade of the tumors. Cadaverine supplementation did not alter the number of primary tumors that grew from the grafted cells (Fig. 1A), but there was a trend towards tumors with lower mass (Fig. IB). In line with that, the number of metastases decreased (Fig.
- Fig. ID Cadaverine induced a mesenchymal-to-epithelial (MET) transition and reduced invasion.
- MET mesenchymal-to-epithelial
- Fig. 3 A Cadaverine treatment changed the fibroblast-like mor phology of the 4T1 cells to a rather cobblestone -like morphology.
- 3C-E show the changes found in CAD-treated 4T1 cells, primary tumors and their metastases from treated mice.
- the effects of CAD were most profound on 4T1 cells but the same trend is evident in vivo: expression of EMT markers MMP2, MMP3, Tgfb3, FgFbpl, Erbb3, Erl, Notchl, IgFbp4, Sppl, Krtl4 and DSC2 are decreased, showing the reversal of the epithelial-mesenchymal transition.
- cadaverine-treated cells were slower in migrating to open areas in scratch as says (Fig. 4A) and also performed worse in Boyden-chamber transmigration tests (Fig. 4B). These data were further supported by the observation that MMP9 expression was suppressed by cadaverine treatment (Fig 4C). Metabolic changes evoked by cadaverine administration were assessed using the Seahorse flux analyzer. Cadaverine treatment reduced glycolytic flux (Fig. 4D) that is a characteristic of breast cancer stroma cells [36]. The“sternness” of 4T1 cells was assessed using the aldefluor assay and a reduction in cancer cell sternness was found (Fig. 4E).
- cadaverine administration was also investigated on five different established breast cancer cell lines of which four were of human (MD -MBA-231, SKBR3, ZR-75-1 and MCF7), while one was of murine origin (4T1).
- the cadaverine concentration used corre sponded to the reference concentration of cadaverine in human serum (100-800 nM) [34, 35, 36].
- cadaverine did not hinder the proliferation of non-transformed primary human skin fibroblasts (Fig. 2A).
- Fig. 2A We assessed whether slower proliferation could be due to the toxicity of cadaverine to cells.
- the proportion of the PI positive cells did not increase upon cadaverine treatment (Fig. 2D), nor did the apoptotic fraction in 4T1 cells (Fig. 2C).
- Cadaverine exerts its beneficial effects through Trace Amino Acid Receptors (TAARs).
- the trace amino ac id receptor family serves as receptors for cadaverine [37, 38, 39]. Indeed, higher expression of TAAR1, TAAR2, TAAR4, TAAR5 and TAAR9 provided better survival in breast cancer (Fig 5A).
- TAAR receptors are G protein-dependent receptors [38] we assessed their involvement by treating 4T1 cells with NF449, a Gsa- subunit-selective G-protein antagonist, a treatment that abolished the anti-EMT effect of cadaverine (Fig. 5B).
- E. coli FdcC levels in the feces of patients having E-cadherin negative breast cancer are lower than in the feces of patients having E-cadherin positive breast cancer.
- Fig. 8 E-cadherin ex pression in breast cancer has been linked to disease progression, metastasis, aggressiveness of the tumor and reduced overall survival [76]. This finding further supports the tumor suppressive role of cadaverine.
- a method for diagnosing early stage breast cancer in a human subject comprising measuring the abundance of at least one bacterium species which is capable of producing cadaverine un der physiological conditions in the human body, in a test sample derived from the subject, wherein the test sam ple comprises human microbiota of said subject, and
- early stage breast cancer refers to stage 0 and stage 1 breast cancer, preferably to stage 0 breast cancer or preferably to stage 1 breast cancer.
- the method is for diagnosing breast cancer stage 0 according to the American Joint Com mittee on Cancer (AJCC) TNM system and the test sample is compared to a reference value typical of the ab sence of stage 0 breast cancer.
- the method is for diagnosing breast cancer stage 1 according to the American Joint Committee on Cancer (AJCC) TNM system and the test sample is compared to a reference value typical of the absence of stage 1 breast cancer.
- the abundance of the bacterium species to be measured is measured by measuring the abundance of the DNA sequence coding for or a gene product of the DNA sequence coding for lysine decar boxylase (LDC).
- the DNA sequence coding for LDC is a DNA sequence in the ldc operon and/or in the cad operon or the gene product is a gene product of a gene of the ldc operon and/or of the cad operon. More preferably, the DNA sequence in the ldc operon is ldcC and/or the DNA sequence in the cad operon is cadA or the gene of the ldc operon is ldcC and/or the gene of the cad operon is cadA.
- the gene product is preferably RNA or protein.
- the protein is preferably LDC, LdcC protein or CadA protein (lysine decarboxylase).
- the RNA is preferably mRNA.
- stage 0 is to be diagnosed, DNA or RNA is used, and when stage 1 is to be disagnosed, protein is used.
- test sample comprising human microbiota of said subject comprises gut microbiota.
- the at least one bacterium species is select ed from Escherichia, Enterobacter and Hafnia.
- the at least one bacterium species is selected from Escherichia coli, Enterobacter cloacae and Hafnia alvei.
- the bacteria to be measured are Escherichia coli, Enterobacter cloacae and Hafnia alvei.
- the abundance of a DNA se quence coding for LDC or a gene product of thereof is measured, wherein the DNA sequence codes for LdcC protein;
- the abundance of a DNA sequence coding for LDC or a gene product of thereof is measured, wherein the DNA sequence codes for LdcC protein and/or CadA protein.
- the test sample may be serum, plasma, whole blood, breast duct fluid, breast tumor tissue or feces.
- the sample in which the abundance of bacteria is to be measured is preferably feces.
- the samples to be compared are corresponding samples, e.g. when the abundance of a bacterium species is measured in a feces sample, the reference value is derived from feces.
- the reference value is a value calculated from samples of individuals not having breast cancer, preferably early stage breast cancer, more preferably stage 0 breast cancer.
- the reference value is calculated from samples of healthy individuals.
- the bacterium species capable of producing cadaverine under physiological conditions in the human body is capable of said production in the human gastrointestinal tract.
- the invention also provides cadaverine for use in the treatment of breast cancer in a patient.
- a method for treating breast cancer in a patient, comprising administering an effective dose of cadaverine to the patient, is also provided.
- the treatment decreases the number of metastases in said patient.
- said treatment decreases the total mass of the primary tumor(s) and/or the total mass of the metastases.
- said treatment decreases the infiltration rate of the primary tumor(s) into surrounding tissues. More preferably, infiltration of the primary tumor(s) into surrounding tissues (e.g. blood vessel or lymphatic vessels) is prevented.
- surrounding tissues e.g. blood vessel or lymphatic vessels
- said treatment reverses endothelial-mesenchymal transition in the breast can cer cells.
- endothelial-mesenchymal transition is indicated and measured by the de creased expression of any one of the following genes in primary tumor tissue or metastasis tissue: MMP2, MMP3, Tgfb3, FgFbpl, Erbb3, Erl, Notchl, IgFbp4, Sppl, Krtl4, DSC2 and combinations thereof.
- MMP3, Erl the expression of any one of the following genes in primary tumor tissue or metastasis tissue is decreased: MMP3, Erl.
- said patient is a human, more preferably a woman.
- cadaverine is administered in a dose providing in the patient a serum concen tration corresponding to the reference physiological serum concentration of cadaverine.
- cadaverine is administered in a dose that provides about 50 nM/1 to 500 mM/l serum concentration in the patient. More preferably, cadaverine is administered in a dose that provides about 100 to 800 nM/1 serum concentration in the patient.
- cadaverine is administered in a dose of about 50 nmol/kg to 500 mM/kg, preferably about 100 to 800 nM/kg or in a dose of 0.1 - 1000 mg daily.
- cadaverine is used to treat early stage breast cancer, in particular stage 0 breast cancer.
- cadaverine is used as an adjuvant therapy, together with a further anti -cancer treatment.
- Designed primers, cadaverine and putrescine were from Sigma-Aldrich (St. Louis, MI, USA). Antibodies were from Cell Signaling Technology (Beverly, MA, USA), Abeam (Cambridge, UK) or from Thermo Fisher Scientific (Rockford, USA) unless otherwise stated.
- 4T1 murine breast cancer cells were maintained in RPMI-1640 (Sigma-Aldrich, R5886) medium containing 10 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine and 1 % pyruvate at 37 °C with 5 % C02.
- MDA-MB-231 and SK-BR-3 human breast cancer cells were maintained in DMEM (Sigma- Aldrich, 1000 mg/1 glucose, D5546) containing 10 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine and 10 mM HEPES at 37 °C with 5 % C02.
- ZR-75-1 human breast cancer cells were maintained in RPMI-1640 (Sigma- Aldrich, R5886) medium con taining 10 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine at 37 °C with 5 % C0 2 .
- MCF-7 human breast cancer cells were maintained in MEM (Sigma-Aldrich, M8042) medium containing 10 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine and 10 mM HEPES at 37 °C with 5 % C0 2 .
- Human primary fibroblast cells were maintained in DMEM (Sigma-Aldrich, 1000 mg/1 glucose, D5546) containing 20 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine and 10 mM HEPES at 37 °C with 5 % C0 2 .
- Cells were seeded in 96-well plates (4T1- 1500 cells/well; MDA-MB-231 and ZR-75-1 - 3000 cells/well; SK-BR-3, MCF-7 and human fibroblast - 5000 cells/well) and were let to attach overnight. Cells were treated with different concentration of cadaverine (Sigma Aldrich, C8561) for 48 hours. After 2 days cells were fixed by the addition of 50 % trichloroacetic acid (TCA, final concentration: 10 %) and the plate was incubated for 1 hour at 4 °C. The plate was then washed 5 times with water and was stained with 0.4 % (w/v) sulphorhodamine B solution in 1 % acetic acid. Unbound dye was removed by washing 5 times with 1 % acetic acid. Bound stain was solubilized with 10 mM Tris base and the absorbance was measured at 540 nm.
- Cells were seeded in 6-well plates (4T1- 750 cells/well; MDA-MB-231, SKBR-3, ZR-75-1 and MCF-7- 1000 cells/well) in complete medium and were treated with the indicated concentrations of cadaverine for 4 days. At the end of the treatment plates were washed in PBS. Colonies were fixed in 4 % PFA for 30 minutes, dried and stained with the solution of May-Griinwald-Giemsa for 30 minutes. Plates were washed with water and the colonies, containing at least 50 cells, were counted using Image J software.
- PI uptake cells were seeded in 6-well plates (MDA-MB-231, ZR-75-1 and MCF-7 - 100,000 cells/well; SKBR-3 and human fibroblast - 200,000 cells/well). After 2 days of cadaverine treatment cells were stained with 100 pg/rnl propidium iodide for 30 min at 37 °C. Supernatant was collected in FACS tubes, cells were washed with PBS and collected in the same FACS tubes (trypsin: PBS 1:1) then samples were analyzed by flow cytometry (FACSCalibur, BD Biosciences).
- ECIS Electric Cell-substrate Impedance Sensing
- ECIS Electro cell-substrate impedance sensing model ZQ, Applied BioPhysics Inc. (Troy, NY, USA) was used to monitor transcellular electric resistance of control and cadaverine treated cells for 20 hours before the treatment, and total impedance values were measured for additional 48 hours upon the indicated cadaverine treatment. Multifrequency measurements were taken at 62.5, 125, 250, 500, 1000, 2000, 4000, 8000, 16000, 32000, 64000 Hz. The reference well was set to a no-cell control with complete medium.
- 4T1 cells were grown on coverslips, and treated with the indicated concentration of cadaverine for 48 hours.
- 5mM NF449 Bio-Techne R&D Systems Kft, 627034-85-9
- G-protein in hibitor was also added to cadaverine-treated cells.
- Cells were washed with PBS, fixed with 4% PFA for 15 minutes and permeabilized using 1 % Triton X-100 for 5 minutes. After washing twice with PBS, cells were blocked with 1 % BSA for one hour at room temperature.
- RNA from cells were prepared using TRIzol reagent (Invitrogen, TR118). 2 pg RNA was reverse transcripted using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA, 4368813) according to the manufacturer’s instructions. qPCRBIO SyGreen Lo-ROX Supermix (PCR
- EMT genes differentially regulated upon cadaverine treatment are listed in Table4. Table 4. List of EMT genes differentially regulated upon cadaverine treatment
- DH5a Escherichia coli were seeded in liquid LB medium. Cells were incubated at 37°C overnight with gen- tie shaking. E. coli cells were then collected with centrifugation and proteins were isolated using RIPA buffer
- RIPA buffer 50 mM Tris, 150 mM NaCl, 0.1 % SDS, 1 % TritonX 100, 0.5 % sodium deoxycolate, 1 mM EDTA, 1 mM Na 3 V0 4 , 1 mM NaF, 1 mM PMSF, protease inhibitor coctail
- Samples were sonicated (Qsonica Q125 Sonicator, Newtown, Connecticut) 3 times for 30 seconds with 50% amplitude.
- Protein extracts were separated on 8% SDS polyacrylamide gels and transferred onto nitrocellulose mem branes by electroblotting. Then membranes were blocked with 5 % BSA, and incubated with anti-Fysine decar boxylase primary antibody (1: 100, Abeam) for overnight at 4 °C. The membranes were washed with IX TBS- TWEEN and incubated with IgG HRP conjugated secondary antibody (1:2000, Cell Signaling Technology).
- 4T1 cells were plated in 6-well plates (150,000 cells/well) and were grown overnight. The plates were man ually scratched with sterile 200 m ⁇ pipette tip, followed by washing the cells with complete growth medium. Then cells were treated with 0.1 mM cadaverine in a 37°C thermostat and were monitored every hour for 2 days using JuLi Br Live cell movie analyzer (NanoEnTek Inc., Seoul, Korea).
- Matrigel invasion assay was carried out on 4T1 cells using Coming BioCoat Matrigel Invasion Chamber (354480). 4T1 cells were seeded in the chambers (50,000 cells/well) in semm free medium, and were grown overnight. Cells were then treated with different concentration of cadaverine (0.1 mM, 0.3 mM, 0.8 mM). The lower chamber contained full 4T1 medium with 100 ng/ml SDFl-alpha (Sigma, SRP4388) as chemoattractant. After 48 hours of cadaverine treatment cells were prepared according to the manufacturer’s instructions and stained with Hematoxylin-Eosin (VWR, 340374T and 341972Q) dye. Cells were then pictured with Opera Phenix High Content Screening System and pictures were analysed using Harmony 4.6 Software. Invasion in dex was calculated from the percentage of invading cells through matrigel membrane and control membrane.
- 4T1 cells were seeded in 96-well Seahorse assay plates (1500 cells/well) and treated with vehicle and cadaverine for 48 hours. Cells were monitored using XF96 oxymeter (Seahorse Biosciences, North Billerica, MA, USA) to measure the changes in oxygen consumption rate (OCR) and in pH (ECAR) after cadaverine treatment similarly to [27]. Data were normalized to protein content.
- Lipid peroxidation was measured by detemining the production rate of thiobarbituric acid reactive substrate (TBARS). 4T1 cells were seeded in T75 flasks and allowed to adhere overnight. Cells were exposed to cadaverine for 48 hours, then collected by centrifugation. 8.1 % SDS, 20 % acetic acid, 0.8 % thiobarbituric acid (TBA) and distilled water was added to the pellet and was heated at 96°C for 1 hour in thermoblock. Sam ples were cooled on ice and centrifugated, the absorbance of the supernatant was measured at 540 nm.
- TBARS thiobarbituric acid reactive substrate
- the level of the enzyme aldehyde dehydrogenase (ALDH) was determined on 4T1 cells using cadaverine treatment. Cells were seeded on 6 well plates (50000 cells/well) and treated with different concentration of cadaverine (0.1 mM, 0.3 mM, 0.8 mM) for 2 days. Cells were then collected and prepared according to the manu facturer’s instructions. We used SKBR-3 cell line for positive control. Changes in the level of ALDH was measured using flow cytometry and the results were analysed with flowing software 2.5.1.
- mice Female mice (4 months of age, 20-25g). Animals were bred in the“specific pathogen free” zone of the Animal Facility at the University of Debrecen, and kept in the“minimal disease” zone during the experiments. 4 mice were housed in one cage (standard block shape 365 c 207 c 140 mm, surface 530 cm 2 ; 1284 L Eurostandard Type II. L from Techniplast). Dark/light cycle was 12 h, and temperature was 22 ⁇ 1°C. Mice had ad libitum access to food and water (sterilized tap water). A total of 32 female mice were used in the study, 16 randomly selected control and 16 cadaverine fed mice. The study was performed in two runs at two different occasions, each run comprising of 8 vehicle -treated and 8 cadaverine-treated mice.
- Tumor was formed in mice by the grafting of 4T1 cells.
- 4T1 cells were suspended (2xl0 6 /ml) in ice cold PBS-matrigel (1: 1, Sigma- Aldrich) at 1: 1 ratio.
- 16 female BALB/c mice received 50 pL injections to the ingui nal fat pads below the lower abdominal nipples on both sides (10 5 cells/injection site).
- Cadaverine stock was prepared in sterilized tap water at lOOx concentration (15 mM) and the stock was stored at -20°C.
- Cadaverine stock was diluted each day to a working concentration of 150 mM in sterile tap water before the treatment.
- Animals received a daily oral dose of 100 m1/30 g body weight from cadaverine solution (8 mice) or vehicle (sterilized tap water, 8 mice).
- researchers administering cadaverine and vehicle solutions were blinded. Treatment was carried out every day during the morning hours between 9am and 11am. Mice were sacrificed on day 14 post grafting.
- “Low infiltration” class means that primer tumor remained in the mammary fat pads without any attachment to muscle tissues. In case the tumor mass attached to the muscle tissue but did not penetrate the abdominal wall, it classi fied as a“medium infiltration” tumor. If the tumor grew into the muscle tissue and totally penetrated the ab dominal wall, it was scored as a“high infiltration” tumor.
- Another cohort was used to assess LdcC protein in the feces of healthy volunteers and breast cancer patients.
- the collection and biobanking of feces was authorized by the Hungarian national authority (ETT).
- Patients and healthy volunteers meeting the following criteria were excluded from the study according to the corresponding national guideline for fecal transplantation [34]: 1) has previous history of breast cancer or had been operated due to neoplasia, 2) has a disease of unknown origin, 3) has chronic contagious disease, 4) had contagious diar rhea 6 months prior to enrollment, 5) taken antibiotics in the 6 months prior to enrollment, 6) had chemothera py, biological therapy or immunosuppressive therapy 6 months prior to enrollment, 7) used intravenous drugs 12 months prior to enrollment, 8) had piercing, tattooing, acupuncture or other endangering behavior or action 12 months prior to enrollment, 9) exposition to an allergen to which the enrolled individual had been sensitized to, 10) undervent colonoscopy 12 months prior to
- the kmplot.com database was used to study the link between gene expression levels and breast cancer sur vival in humans.
- the association of known mutations with breast cancer was retrieved from www.intogen.org/.
- the sequence of the CadA and LdcC ORFs were retrieved from the KEGG (www.genome.jp/kegg/) database.
- cacccagcgc atattcaaag aag 23 ⁇ 210> 5
- NCBI_GEO_Profiles LDC expression in epithelium and stroma of normal breast and invasive breast cancer.
- NCBI_GEO_Profiles Normal epithelium vs. breast cancer epithelium in patients.
- CSCs cancer stem cells
- the Escherichia coli ldcC gene encodes another lysinedecarboxylase, probably a constitutive enzyme. Genes Genet. Syst. (1997) 72, p. 167-172.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of diagnosis and treatment of early stage breast cancer. Specifically, a method is provided for determining whether a subject has early stage breast cancer by measuring the abundance of a bacterium species comprising a DNA sequence coding for a lysine decarboxylase. A method for mitigating breast cancer initiation and/or promotion and/or progression in a subject is also provided.
Description
Treatment and diagnosis of breast cancer
FIELD OF THE INVENTION
The invention relates to the field of diagnosis and treatment of early stage breast cancer. Specifically, a method is provided for determining whether a subject has early stage breast cancer by measuring the abundance of a bacterium species comprising a DNA sequence coding for a lysine decarboxylase. A method for mitigating breast cancer initiation and/or promotion and/or progression in a subject is also provided.
BACKGROUND OF THE INVENTION
Microbes that live on the surface or the cavities of the human body affect a large set of pathophysiological processes ranging from metabolic diseases to psychiatric disorders [1-4] or neoplastic dieases [3, 5-7]. The number of directly tumorigenic bacteria is extremely low (-10 species) [8], however, dysbiosis is associated with cancers of the urinary tract [9], cervix [10], skin [11], airways [12], the colon [13], lymphomas [14, 15], prostate [9] and breast cancer [16-23]. Dysbiosis is often reflected as a loss of diversity of the microbiota (e.g.
[17]). In colon carcinogenesis, microbes probably promote the malignancy. However, the majority of the afore mentioned cancers are located distantly from larger depots of microbes, hence, suggesting indirect induction or promotion mechanisms. Indeed, bacterial metabolites emerge as“endocrine” agents that are produced by the microbiome, are absorbed into the circulation, and exert their biological effects distantly
Cadaverine (CAD; pentane- 1,5 -diamine) is produced by the decarboxylation of lysine that is performed by lysine decarboxylase (LDC) enzymes. Human cells code and express LDC and numerous bacterial species of the human microbiome also express LDC either in a constant (LdcC in the LDC operon) or in an inducible (CadA in the Cad operon) fashion [32, 33]. Bacteria use diamines, like cadaverine or putrescine, generated by the decarboxylation of lysine or arginine, to buffer the pH of their environment [28]. The effects of cadaverine on cancer cells and its role in carcinogenesis are not yet characterized in detail.
Loser et al. [35, 36] found that cadaverine concentrations in the pancreatic tissue of patients with pancreatic cancer were elevated. Depierre et al. [62] also found that the excretion (in urine) of cadaverine was increased versus controls in lung cancer. US20170016901A1 describes that elevated levels of polyamines (among them cadaverine) may indicate the presence of breast cancer. EP3064940 also shows that the concentration of cadaverine measured in the saliva of breast cancer patients is higher than that of the controls.
WO2013045826A1 describes that a mixture depleted of cadaverine, putrescine, spermidine and spermine and on the other hand containing agmatine at a concentration higher than that supplied by an average food ration is beneficial in the treatment of pathologies associated with cellular hyperproliferation.
Breast cancer is among the most common cancers worldwide. Early detection may be a clue for efficient therapy without irreversible damages.
BRIEF DESCRIPTION OF THE INVENTION
The invention prvides a method for determining whether a subject has early stage breast cancer or has an in creased probability of having early stage breast cancer by measuring, in a sample derived from said subject, the level of bacterial cadaverine synthesis.
The invention provides a method for determining whether a subject has early stage breast cancer or has an increased probability of having early stage breast cancer by measuring the abundance of a bacterium species comprising a DNA sequence coding for a lysine decarboxylase (LDC), the level of a DNA sequence coding for an LDC of a bacterium species, or the level of a gene product of a DNA sequence coding for an LDC produced by a bacterium species, in a sample derived from the subject, wherein the sample comprises microbiota from the subject and wherein the bacterium species is part of the microbiota of the subject.
The invention also provides a method for determining whether a subject is at an increased risk of developing breast cancer, by measuring the abundance of a bacterium species comprising a DNA sequence coding for a lysine decarboxylase (LDC), the level of a DNA sequence coding for an LDC of a bacterium species, or the level of a gene product of a DNA sequence coding for an LDC produced by a bacterium species, in a sample derived from the subject, wherein the sample comprises microbiota from the subject and wherein the bacterium species is part of the microbiota of the subject. The invention also provides cadaverine for use in the treatment or prevention of breast cancer in a subject and cadaverine for use in mitigating cancer initiation and/or promo tion and/or progression in a subject. The invention is further detailed in the following paragraphs.
A method for determining whether a subject
i) has early stage breast cancer or
ii) has an increased probability of having early stage breast cancer or
iii) is at an increased risk of developing breast cancer,
the method comprising
assessing, in a test sample from the subject, the level of bacterial cadaverine synthesis, the sample comprising microbiota from the subject or
measuring the level of cadaverine in a test sample from the subject
wherein
a decreased level of bacterial cadaverine synthesis or
a decreased level of cadaverine, respectively,
indicates that the subject has early stage breast cancer or an increased probability of having early stage breast cancer or is at an increased risk of developing breast cancer.
Preferably, the level of bacterial cadaverine synthesis is assessed by measuring
the abundance of a bacterium species comprising a DNA sequence coding for a lysine decarboxylase (LDC) or
the level of a DNA sequence coding for an LDC of a bacterium species, or
the level of a gene product of a DNA sequence coding for an LDC produced by a bacterium species, wherein
a lower abundance of the bacterium species in the test sample or
a lower level of the DNA sequence coding for an LDC of a bacterium species, or
a lower level of the gene product of a DNA sequence coding for an LDC produced by a bacterium spe cies, respectively,
compared to the corresponding reference value
indicates that the subject has early stage breast cancer or an increased probability of having early stage breast or is at an increased risk of developing breast cancer.
Preferably the breast cancer is stage 0 or 1 breast cancer according to the American Joint Committee on Cancer (AJCC) TNM system. Preferably, the cancer is stage 0 breast cancer and the test sample is compared to a reference value typical of the absence of stage 0 breast cancer. Alternatively, the cancer is breast cancer stage 1 the test sample is compared to a reference value typical of the absence of stage 1 breast cancer.
Preferably the DNA sequence coding for LDC is ldcC or cadA.
Preferably the test sample is a feces sample.
Preferably the bacterium species is selected from Escherichia, Enterobacter and Hafnia. More preferably the bacterium species is selected from Escherichia cob, Enterobacter cloacae and Hafnia alvei.
In a preferred embodiment
a lower abundance of the bacterium species in the test sample or
a lower level of the DNA sequence coding for an LDC of a bacterium species
compared to the corresponding reference value
indicates that the subject has stage 0 breast cancer or an increased probability of having stage 0 breast cancer or is at an increased risk of developing breast cancer.
In a more preferred embodiment a lower level of the DNA sequence coding for an LDC of a bacterium spe cies
compared to the corresponding reference value
indicates that the subject has stage 0 breast cancer or an increased probability of having stage 0 breast cancer or is at an increased risk of developing breast cancer.
In a preferred embodiment
a lower level of the gene product of a DNA sequence coding for an LDC produced by a bacterium species compared to the corresponding reference value
indicates that the subject has stage 1 breast cancer or an increased probability of having stage 1 breast cancer or is at an increased risk cancer of developing breast cancer. Preferably the gene product is LDC protein.
Preferably the subject is a human, preferably a woman, more preferably a postmenopausal woman.
In another aspect of the invention cadaverine for use in the treatment or prevention of breast cancer in a sub ject is provided.
Also cadaverine for use in mitigating cancer initiation and/or promotion and/or progression in a subject is provided.
Preferably the subject
has early stage breast cancer or
has an increased probability of having early stage breast cancer or is at an increased risk of developing breast cancer. More preferably the subject has early stage breast cancer.
Preferably prior to said use
the abundance of a bacterium species comprising a DNA sequence coding for LDC,
the level of a DNA sequence coding for an LDC of a bacterium species, or
the level of a gene product of a DNA sequence coding for an LDC produced by a bacterium species, is lower in a test sample comprising microbiota from said subject than the corresponding reference value or the level of cadaverine in a test sample from the subject is lower than the corresponding reference value..
In a preferred embodiment the cadaverine for use is for use in
decreasing the infiltration rate of primary tumor(s) into surrounding tissues and/or
decreasing the number of metastases and/or
decreasing the total mass of the primary tumor(s) and/or
decreasing the total mass of the metastases.
Preferably the test sample is feces. Preferably the bacterium species is selected from Escherichia, Enterobacter and Hafiiia. More preferably the bacterium species is selected from Escherichia coli, Enterobacter cloacae and Hafiiia alvei. Preferably the DNA sequence coding for an LDC is cadA or ldcC. Preferably the gene product is LDC protein.
In another aspect the invention provides a method for mitigating breast cancer initiation and/or promotion and/or progression in a subject, comprising
measuring in a test sample comprising microbiota from said subject
the abundance of a bacterium species comprising a DNA sequence coding for a lysine decarboxylase (LDC) or
the level of a DNA sequence coding for an LDC of a bacterium species, or
the level of a gene product of a DNA sequence coding for an LDC produced by a bacterium species, or measuring the level of cadaverine in a test sample from said subject,
administering cadaverine to the subject to provide or restore physiological serum concentration of cadaverine if
the abundance of the bacterium species comprising a DNA sequence coding for LDC or
the level of the DNA sequence coding for an LDC of a bacterium species or
the level of the gene product of a DNA sequence coding for an LDC produced by a bacterium species, or the level of cadaverine, respectively,
is lower than the corresponding reference value, and
monitoring the presence of breast cancer in the subject and/or the serum cadaverine level of said subject, and if necessary, administering another anticancer therapy together or without the administration of cadaverine.
In a preferred embodiment the subject has early stage breast cancer. Preferably the test sample is feces. Pref erably the bacterium species is selected from Escherichia, Enterobacter and Hafnia. More preferably the bacte rium species is selected from Escherichia coli, Enterobacter cloacae and Hafnia alvei. Preferably the DNA sequence coding for an LDC is cadA or ldcC. Preferably the gene product is LDC protein.
In another aspect a pharmaceutical composition comprising cadaverine and at least one pharmaceutically ac ceptable excipient or carrier is provided. Preferably the pharmaceutical composition is for use in mitigating breast cancer initiation and/or promotion and/or progression. Preferably the the pharmaceutical composition is administered to provide or restore physiological serum concentration of cadaverine in a subject having early stage breast cancer or a lower serum cadaverine level compared to the physiological serum cadaverine.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Cadaverine treatment reduces breast cancer aggressiveness in vivo. Female Balb/c mice were grafted with 4T1 cells as described in the Examples and were treated with cadaverine (CAD, 500 nmol/kg q.d. p.o.) or vehicle (VEH, CTL) (n=16/16) for 16 days before sacrifice. For pathological measurements 6 samples from VEH group and 8 samples from cadaverine group were analysed. In VEH and CAD-treated mice (A) the number and (B) mass of primary tumors were counted and the (C) number and (D) mass of metastases were measured upon autopsy. (E) Upon autopsy, the infiltration rate of the primary tumor was scored. Significant level (p=0.002) was calculated using Fisher's exact test with 2x3 Contingency Table. (F-I) Primary tumors were formalin-fixed and were embedded into paraffin, then sections were hematoxylin-eosin stained and were scored for (F) mitosis, (G) mitosis/hpf, (H) nuclear pleomorphism.
Data is plotted as mean ± SEM. In box-whiskers charts (panels B and D) middle lines indicate the median, while squares sign the mean of data. A-D: two-tailed Student t-test. * and ** indicate p<0.05 and p<0.01, re spectively.
Figure 2. Cadaverine reduces the proliferation and colony forming ability of breast cancer cells. (A) 4T1 (n=6 in octuplicates), MDA-MB-231 (n=3 in octuplicates), SKBR-3 (n=3 in octuplicates), ZR-75-1 (n=3 in octuplicates) and MCF-7 (n=3 in octuplicates) breast cancer cells and primary fibroblasts cells were treated with cadaverine in the concentrations indicated for 48 hours then total protein concentrations were determined in SRB (sulphorhodamine B) assay. Values are expressed as fold changes, where 1.0 means protein content in the control cells (indicated by a dotted line). (B) 4T1, MDA-MB-231, SKBR-3, ZR-75-1 and MCF-7 (n=3 for each in one replicate) cells were treated with cadaverine in the concentrations indicated for 4 days. Colonies were then stained according to May-Griinwald-Giemsa and counted using Image J software (n=3). (C) 4T1 cells (n=3 in triplicates) were treated with cadaverine in the concentrations indicated in the examples for 48 hours. Cells were stained with Annexin-FITC-PI Apoptosis Kit and analyzed by flow cytometry (n=4). (D) 4T1 (n=3 in triplicates), MDA-MB-231 (n=4 in triplicates), SKBR-3(n=4 in triplicates), ZR-75-1 (n=3 in triplicates) and MCF-7 (n=3 in triplicates) were treated with cadaverine in the concentrations indicated in the examples for 48 hours. Dead cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Data is plotted as mean ± SEM.
Figure 3. Cadaverine treatment reverses EMT (epithelial-mesenchymal transition) of breast cancer cells. (A- B) In control and cadaverine-treated 4T1 cells (A) total cellular impedance was measured by ECIS (electric cell- substrate impedance sensing) (n=2 in duplicate) (p=0.0484 at 36 hrs and 0.0487 at 40 hrs) and (B) morphology of the actin cytoskeleton (representative picture of control and 0.1 mM cadaverin-treated cells) was assessed after Texas Red-X Phallodin+To-Pro-3 staining (n=2 in triplicates), ratio (%) of epithelial and mesenchymal cells was shown on bar chart. Significance was calculated using Chi-square test in Microsoft Excel (p<0.001 between control and 0.1 mM cadaverine, p<0.001 between control and 0.3 mM cadaverine, p<0.001 between control and 0.8 mM cadaverine). (C-E) Expression of a set of genes involved in EMT were assessed by RT- qPCR in (C) 4T1 cells (n=3), (D) primary tumors (n=16/16) and in (E) metastases (n=16/16). All data is ex pressed as fold change. The line that equals to 1 (no change) indicates the average of the control samples. Data is plotted as mean ± SEM. On panel A significance was calculated using One-way Anova. On panels C-E sig-
nificance was calculated using two-sample student t-test, (two-tailed), while on panel B One-way Anova was used to calculate differences. *, ** and *** p<0.05, p<0.01 or p<0.001 respectively.
Figure 4. Cadaverine treatment attenuates movement, invasion ability, mitochondrial oxidation and sternness of 4T1 cells. (A) 4T1 cells were treated with cadaverine in the concentration indicated for 48 hours after scratching a 4T1 cell layer. Subsequent closure of the wound was assessed by the JULI-Br live cell analyzer system (n=2). Significance was calculated using two-sample student t-test (two-tailed). (B) 4T1 cells were treat ed with cadaverine in the concentration indicated for 48 hours and subsequently invasion capacity of the cells was measured using the Corning matrigel invasion chamber. Cells were counted using the Opera HCS system and invasion index was calculated. (p=0.003 both between control and 0.1 mM cadaverine, and control and 0.3 mM cadaverine, p= 4.14 x 10 4 between control and 0.8 mM cadaverine). (C) 4T1 cells were treated with vehicle and cadaverine for 48 hours, then cells were stained for MMP9 and nucleus (DAPI) and sections were analysed by confocal microscopy using a Leica SP8 confocal system. MMP9 content was calculated from the total cellu lar fluorescence masured by the Image J software (p=0.001 between both control and 0.3 mM cadaverine and control and 0.8 mM cadaverine). (D) 4T1 cells were treated with vehicle and cadaverine for 48 hours (n=3 in 24 replicates), then cells were subjected to a Seahorse XF96 analysis. Oxygen consumption rate (OCR) and extra cellular acidification rate (ECAR) were measured and plotted (pOCR=0.006 between control and 0.1 mM cadaverine, pOCR p<0.001 between control and 0.3 mM cadaverine, pOCR=1.8 x 10 5 between control and 0.8 mM cadaverine). (E) 4T1 cells were treated with vehicle and cadaverine in the concentration indicated for 48 hours (n=3 in triplicates), then cells were subjected to an Aldefluor assay (p=0.035 between control and 0.3mM cadaverine). Data is plotted as mean ± SEM. Significance was calculated using One-way Anova. *, ** and *** indicate p<0.05, p<0.01 and p<0.001, respectively.
Figure 5. TAARs are seemed to be responsible for the effect of cadaverine. (A) Patient data was accessed at kmplot.com. Kaplan-Meier plots show the correlation between the mRNA expression of human TAARs and survival in breast cancer. (B) 4T1 cells were treated with, 100 nM cadaverine (48 hours) or 100 nM cadaverine (48 hours) in combination with 5 mM NF449 G protein inhibitor (last 48 hours of cadaverine treatment), cells were stained with Texas Red-X Phallodin- and To-Pro-3 then cells were assessed by confocal microscopy (n=2 in triplicates). Ratio (%) of epithelial and mesenchymal cells was shown on bar chart (cells were treated with cadaverine in the indicated concentrations in combination with 5 mM NF449G protein inhibitor). Data is plotted as mean ± SEM.
Figure 6. Cadaverine biosynthesis is suppressed in early stages of breast cancer. (A) Fluman fecal DNA samples were collected from 48 patients with different stages of breast cancer, and from 48 healthy patients. The abundance of DNA coding for CadA and LdC of the indicated bacterial species were determined in the fecal DNA samples by RT-qPCR. Median values are indicated by a line. (B) Fluman fecal samples were collected from stage 1 breast cancer patients (n=7) and from healthy (control) subjects (n=3). The E. coli LdcC protein level was determined using Western blot. Flereby we show a representative image. Band intensity was normal ized to total protein content assessed by ponceau-S staining of whole blots. Box chart of LDC protein expres sion from fecal samples. The middle line indicates the median, while squares sign the mean of data. Data was normalized to the mean of control samples. Blots were routinely cut as the representative blot. (C-D) Patient
data was accessed at kmplot.com. Kaplan-Meier plots the correlation between the mRNA expression of human LDC and survival in breast cancer. Graphs show the correlation between LDC and disease survival in different forms and stages of the disease. Those arrays were also included where ER status was deducted from gene ex pression data. ** indicates statistically significant difference between control and stage 1 breast cancer treated groups at p<0.01. Significance was calculated using two-sample student t-test.
Figure 7. Cadaverine reverts EMT in breast cancer cell lines. (A-B) In control and cadaverine-treated (A) MDA-MB-231 (n=l in triplicates) and (B) SKBR-3 (n=l in triplicates) breast cancer cells morphology of the actin cytoskeleton was assessed after Texas Red-X Phallodin+To-Pro-3 staining. Ratio (%) of epithelial and mesenchymal cells was shown on bar charts. Significance was calculated using Chi-square test in Microsoft Excel.
Figure 8. Fecal E. coli LdcC expression was determined from total fecal extracts by Western blotting (n=29/5) from breast cancer patients. LdcC signal was normalized for total protein (Amido black signal from the membrane).
DETAILED DESCRIPTION OF THE INVENTION
Bacterial cadaverine biosynthesis has been found to be suppressed in breast cancer. Bacteria of the human microbiota capable of producing cadaverine may be used as an early indicator of the presence or risk of breast cancer.“Bacterial cadaverine synthesis” refers to the potential of the microbiota to produce cadaverine, prefera bly by the enzymatic decarboxylation of lysine. The level of“bacterial cadaverine synthesis” may depend on the number of bacteria capable of producing cadaverine, e.g the number of bacteria comprising a coding sequence for a lysine decarboxylase enzyme or the rate of expression of the enzyme or the level of activity of the enzyme or the level of the enzyme. Bacteria of the gut capable of producing cadaverine are less abundant in the feces of patients with early stage breast cancer. For example, the concentrations or amounts of LDC DNA, mRNA or protein are lower in the feces sample of an individual with stage 0 or 1 breast cancer than in a control feces sample. The“stage” of a cancer in this description is to be understood as a stage determined using the American Joint Committee on Cancer (AJCC) TNM system staging. American Joint Committee on Cancer (AJCC) (TNM system) staging system was used according to the 7th edition. The term“early stage” refers to stage 0 and stage 1, preferably to stage 0 or preferably to stage 1. Accordingly, the invention provides a method for diagnosing breast cancer by measuring the abundance of at least one bacterium species which is capable of producing cadaverine under physiological conditions in the human body, in a test sample derived from the subject, wherein the test sample comprises microbiota of said subject. The term“abundance” or“level” in general is meant as a proportion of a given specimen in a given pool relative to certain similar specimens. The term“abundance” or “level” may refer to a concentration or quantity of the specimen. The“abundance” of a bacterium species may refer to the concentration or the number of cells measured in a sample (e.g. a fecal sample). The term“abun dance of the DNA sequence coding for lysine decarboxylase (LDC) relates to the proportion of the special seg ment of bacterial DNA making up the DNA sequence coding for lysine decarboxylase in a DNA pool or isolate (e.g. total DNA in a sample or a pool of samples). The term“abundance (or level) of the DNA sequence coding for lysine decarboxylase” may relate to the amount of the special segment of bacterial DNA making up the
DNA sequence coding for lysine decarboxylase in a sample (test sample or reference sample). The abundance or level of an RNA or protein molecule is defined accordingly, i.e. it relates to the amount of the RNA or protein in the sample. In the context of the specification,“abundance” may refer to relative abundance, e.g. the abundance of E. coli in a sample of a patient may be calculated based on the abundance of E. coli in a reference sample. Cadaverine is produced by direct decarboxylation of L-lysine catalyzed by lysine decarboxylase. In the context of the invention a bacterium is capable of producing cadaverine if the genome of said bacterium comprises a DNA sequence coding for a lysine decarboxylase. Particularly, a bacterium is capable of producing cadaverine if its genome comprises a cad and/or ldc operon, more particularly cadA and/or ldcC. More particularly, a bacte rium is capable of producing cadaverine if it expresses a lysine decarboxylase. In the context of the invention a bacterium that is capable of producing cadaverine produces cadaverine by the decarboxylation of lysine which is catalyzed by LCD. Preferably, a bacterium that is capable of producing cadaverine under physiological condi tions in the human body, is capable of producing cadaverine in the gastrointestinal system. The bacterium whose abundance is to be measured is a bacterium that is part of the healthy human microbiota, preferably the gut microbiota, preferably the fecal microbiota, more preferably the gut and the fecal microbiota.
The DNA sequence coding for a lysine decarboxylase may be a constitutive (e.g. ldcC) or an inducible gene (e.g. cadA) and the expression of the protein may be constitutive or inducible. The capability of a bacterial spe cies/strain/genus to convert lysine to cadaverine under physiological conditions may be tested in vitro. In an appropriate in vitro assay bacterial lysates and lysine are mixed and at the end of the assay cadaverine is detect ed by e.g. mass spectrometry and the lysine/cadaverine ratio is calculated. Lysine carboxylase activity may be measured as described in Inoue et al. Although culturing (gut) microbes has its challenges, the skilled person will find guidance in e.g. Sommer [68]; Lagier et al. [69]; Browne et al [70]; Yamamoto et al. [70]. To mimic the natural growing conditions of the gut microbiota, one might use specialized testing conditions, e.g the i- screen platform by TNO (The Netherlands Organization for Applied Scientific Research). To determine whether a bacterium species is (capable of) coexisting with a human subject or is capable to grow in the gut, one might consult and search sequence databases such as those of the NIH Human Microbiome Project (https://commonfund. nih.gov/hmp; httt>s://t>ortal.hmt>dacc.org/ as of 19 July 2018) or the Human Pan-Microbe Communities (http://www.hpmcd.org/ as of 19 July 2018). It is also possible to sequence and compare a charac teristic genetic portion of a bacterium of interest with reference samples of healthy humans. The methods for measuring the (relative) quantity of bacteria, DNA, RNA or protein are well known to the skilled person and include e.g. quantitative real time PCR assays, (faecal) immunochemical tests, Western blot, turbidimetry, anti body and mass spectrometry-based assays, RT -coupled new generation sequencing. Guidance is also found in the Examples, and in e.g. Liang et al [63]; Wong SH, et al. [64] ; E. Nabizadeh et al. [65]; Goedert et al. [66]; Xie et al. [67]. A“decreased level of bacterial cadaverine synthesis” in a test sample refers to a decrease of bacterial cadaverine synthesis as compared to a reference level. The (corresponding) reference value of abun dance or quantity or level or concentration may be derived from a plurality of samples from individuals not having early stage breast cancer, preferably not having stage 0 or stage 1 breast cancer, preferably not having cancer and most preferably from healthy individuals. E.g. the corresponding reference value of the serum cadaverine concentration may be the physiological serum concentration of cadaverine. Likewise, in the case of
the level of a DNA sequence coding for an LDC of a bacterium species, the corresponding reference value is the value derived from measurements of the level of the DNA sequence coding for the (same) LDC of the (same) bacterium species in the same type of samples from individuals not having early stage breast cancer, preferably from healthy individuals. Accordingly, a level or concentration of an LDC protein in a test sample is compared to the characteristic value derived from measurements of the level or concentration of the LDC protein in the same type of samples from individuals not having early stage breast cancer, preferably from healthy individuals.
It is also possible to search appropriate databases for bacteria known to be present in the human body, pref erably in the human gastrointestinal tract, preferably in the intestine, wherein a DNA sequence coding for a LDC was annoted. Accordingly, a screening method is provided, wherein bacteria suitable to indicate the pres ence of early stage breast cancer may be screened for. In the screening method an appropriate database is searched for bacteria wherein a DNA sequence coding for a LDC has been annoted. By measuring the abun dance of such bacteria in healthy individuals and early stage breast cancer patients, bacteria suitable to indicate the presence of early stage breast cancer may be identified. Such databases (e.g. the Human Pan-Microbe Com munities (HPMC) database, the NIH Human Microbiome Project, the Integrated reference catalog of the human gut microbiome) are well-known to the skilled person, and may be found in e.g. Turnbaugh et al. [71] The hu man microbiome project: exploring the microbial part of ourselves in a changing world. Nature 2007; 449(7164): 804-810, e.g the KEGG database.
The term“human microbiota” refers to the microbes capable of living in or on the human body. The terms “(human)”“gut microbiota” and“gut microbiome” refer to species of the (human) microbiota living in the (hu man) gastrointestinal tract and the term“fecal microbiota” refers to microbes found in the feces. The term “microbiome” and“microbiota” as used in the description may refer to both the (human) microbiota and (hu man) microbiome and the gut/fecal microbiota and gut/fecal microbiome, preferably to the gut/fecal microbiota and microbiome. Bacteria capable of producing cadaverine may reside in the gut, on the skin or in other tissues, such as breast tissue and in the feces.
The abundance of the DNA coding for LdcC and CadA was assessed in human fecal DNA from the experi mental cohort described in the Examples. The abundance of Escherichia coli CadA and also E. coli, Enterobacter cloacae and Hafnia alvelii LdcC DNA in breast cancer patients was decreased compared to healthy individuals (Fig. 6A). Decreased cadA and ldcC abundance was more pronounced in clinical stage 0 patients as compared to the pool of all patients (Fig. 6A). In the feces of stage 1 patients E. coli LdcC protein levels were markedly lower than the levels in the feces of healthy subjects (Fig. 6B).
The GEO database has been assessed to study LDC expression in human breast cancer. There was no differ ence in LDC mRNA expression between control and breast cancer cases [39-42] or in LDC expression of the normal breast epithelium and cancer epithelium in patients [42, 43]. Rather as an exception, LDC expression was lower in basal-like breast cancer as compared to control (normal) breast epithelium of non-diseased indi viduals [43, 44].
The kmplot.com database was used to assess how expression of LDC in humans affects the outcome of breast cancer. Differences in LDC expression did affect overall survival of the patients, in grade 1 patients high er expression of LDC was associated with significantly longer survival than lower expression of LDC (Fig. 6C).
Interestingly, while LDC expression did not affect survival in ER- PR- patients, higher LDC expression corre lated with better survival in ER+ PR+ patients (Fig. 6D). High expression of human LDC prolongs survival in early stage breast cancer patients, supporting the potential anti-cancer properties of cadaverine.
It has been also found that cadaverine administered in a dose corresponding to the physiological serum con centration in humans exerted antitumor effects in mice grafted with either human -derived or mouse-derived breast cancer cells. The physiological concentration in humans refers to a reference range measured in healthy (i.e. not having breast cancer) individuals and is considered to be 100-800 nM/1 (in the serum) [34, 35]. There fore, the invention provides cadaverine for use in the treatment or prevention of breast cancer in a subject, cadaverine for use in mitigating cancer initiation and/or promotion and/or progression in a subject and cadaverine for use in the treatment or prevention of breast cancer in a subject or in mitigating cancer initiation and/or promotion and/or progression in a subject, wherein in said use physiological serum concentration of cadaverine is provided or restored in said subject. In the context of said use or said cadaverine for use the term “physiological serum concentration” refers to the serum concentration (or a range of concentrations) measured in healthy subjects. Cadaverine plays a tumor suppressor role in breast cancer, in concentrations corresponding to the human reference range. Moreover, cadaverine, in the concentrations corresponding to its reference con centration, did not have a cytostatic effect on primary fibroblasts, suggesting that these effects are specific for tumor cells. Cadaverine exerts its effects through inhibiting EMT, cellular movement, chemotaxis and metasta sis. It has been also found that cadaverine treatment reduces the aggressivity of breast cancer, as indicated by the decreased rate of mitosis and heterogeneity of nuclear morphology, reduced number of metastases, lower mass and infiltration of the primary tumor. In human patients, rate of mitosis and heterogeneity of nuclear mor phology are part of the Nottingham grading system. Our results suggest that the effect of cadaverine treatment may be detected by the repeated grading of the tumor. Change in nuclear score due to cadaverine treatment is shown on Fig. 1H.
Infiltration rate of the primary tumors has been found to be lower after cadaverine treatment (Fig. IE). In our experiments“low infiltration” class means that the primeary tumor remained in the mammary fat pads without any attachment to muscle tissues. In case the tumor mass attached to the muscle tissue but did not penetrate to the abdominal wall, it was classified as a“medium infiltration” tumor. If the tumor grew into the muscle tissue and totally penetrated the abdominal wall, it was scored as a“high infiltration” tumor. In the clinics, histopathological evaluation may be used to determine infiltration rate, e.g. sample specimen after surgery or biopsy may be used. Breast cancer is considered systemic when the tumor has infiltrated blood or lymphatic vessels.
Besides the total mass of the primary tumor(s), also the total mass of the metastases decreased after cadaverine treatment in our experiments. In human patients, e.g. FDG-PET may be used to follow such changes. FDG-PET is also suitable to calculate the number of metastases.
The dose of cadaverine used in the experiments with mice corresponds to the physiological concentration in the serum of healthy humans. The human dose of cadaverine to be used may be calculated by known standards, e.g. the Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Thera peutics in Adult Healthy Volunteers from the U.S. Department of Health and Human Services Food and Drug
Administration Center for Drug Evaluation and Research (CDER). Cadaverin may be administered in a dose of about 50 nmol/kg bodyweight to 500 mihoΐ/kg bodyweight, preferably about 50 nmol/kg bodyweight to 250 pmol/kg bodyweight, preferably about 50 nmol/kg bodyweight to 100 pmol/kg bodyweight, preferably about 100 nmol/kg bodyweight to 800 nmol/kg bodyweight, or preferably in a dose providing a serum concentration of 50-2000 nM/1 preferably 100-800 nM/1, more preferably 100-500 nM/1 or 50-500 nM/1.
In a preferred embodiment cadaverine is administered in a dose restoring physiological serum concentration of cadaverine. The term“physiological serum concentration of cadaverine” refers to a reference serum concen tration measured in healthy subjects. Loser et al found the serum concentration of cadaverine to be 0.32 (±SEM:0.07) nmol/ml in healthy volunteers. In an embodiment “physiological serum concentration of cadaverine” refers to a serum concentration of from about 0.15 nmol/ml to about 0.6 nmol/ml. In a preferred embodiment the“physiological serum concentration of cadaverine” refers to a serum concentration of from about 20 nmol/ml to about 0.5 nmol/ml from about 25 nmol/ml to about 0.4 nmol/ml. In a study by the Europe an Food Safety Authority (Scientific opinion on risk based control of biogenic amine formation in fermented foods EFSA Journal 2011;9(10):2393), cumulative daily exposure to cadaverine was found to be between 52.5 and 100.8 mg/day in European countries. In an in vitro cytotoxicity study on human cells (del Rio et al. The biogenic amines putrescine and cadaverine show in vitro cytotoxicity at concentrations that can be found in foods www.nature.com/scientific reports (2019) 9: 120) non-observed adverse effect level (NOAEL) for cadaverine was found to be 2.5 mM (equivalent to 255.45 mg/kg), while the lowest observed adverse effect level (LOAEL) found to be 5 mM (equivalent to 510.89 mg/kg). The IC50 value for cadaverine was 40.72 ± 1.98 mM. The skilled person may calculate a safe dose for cadaverine from these data.
Cadaverine may be administered in the form of a pharmaceutical composition, wherein the pharmaceutical composition comprises cadaverine and at least one pharmaceutically acceptable excipient or carrier. Cadaverine is a liquid on room temperature, which fumes and attracts CO2 from the air. Cadaverine is soluble in water and alcohol, however, incompatible with acid chlorides, acids, acid anhydrides, strong oxidizing agents, carbon dioxide. These properties must be considered when manufacturing an appropriate dosage form.The term“miti gating” refers to delaying, inhibiting, decreasing any of the processes leading to and playing a role in cancer development, including the processes in which cells change to grow and divide continously, spread and invade other tissues.
The term“primary tumor” is used in the generally accepted meaning thereof, indicating the original, or first, tumor(s) in the body. Cancer cells from a primary tumor may spread to other parts of the body and form new, or secondary, tumors.
Cadaverine teatment (500 nmol/kg) decreased the invasivity of the primary tumors in mice homotopically grafted with 4T1 breast cancer cells (Fig. IE). Histological examination of the primary tumors revealed that cadaverine teatment decreased the rate of mitosis (Fig. 1F-G), the heterogeneity of nuclear morphology (Fig. 1H) and the pathological grade of the tumors. Cadaverine supplementation did not alter the number of primary tumors that grew from the grafted cells (Fig. 1A), but there was a trend towards tumors with lower mass (Fig. IB). In line with that, the number of metastases decreased (Fig. 1C) and, as with the primary tumors, there was a trend for smaller tumors in the cadaverine-teated mice (Fig. ID).
Cadaverine induced a mesenchymal-to-epithelial (MET) transition and reduced invasion. Better adherence of mesenchymal-like cancer cells was found in the ECIS (electric cell-substrate impedance sensing) system, in which 0.1 mM cadaverine increased resistance (Fig. 3 A). Cadaverine treatment changed the fibroblast-like mor phology of the 4T1 cells to a rather cobblestone -like morphology (Fig. 3B) that is characteristic for epithelial cells [27], as shown by phallodin-Texas Red staining to visualize the arrangement of the actin cytoskeleton. Treatment of MDA-MB-231, SKBR-3 and ZR-75-1 breast cancer cell lines with cadaverine led to similar mor phological changes. RT-qPCR screen on EMT genes revealed differential expression of genes after cadaverine treatment (Table 4). MMP2, MMP3 and MMP9 and support movement; Tgfb3, FgFbpl, Erbb3 and Erl support proliferation; while Krtl4, Notchl, CDH1, IgFbp4 and Sppl support cell adhesion (Fig. 3D-E). Fig. 3C-E show the changes found in CAD-treated 4T1 cells, primary tumors and their metastases from treated mice. The effects of CAD were most profound on 4T1 cells but the same trend is evident in vivo: expression of EMT markers MMP2, MMP3, Tgfb3, FgFbpl, Erbb3, Erl, Notchl, IgFbp4, Sppl, Krtl4 and DSC2 are decreased, showing the reversal of the epithelial-mesenchymal transition.
In line with these observations, cadaverine-treated cells were slower in migrating to open areas in scratch as says (Fig. 4A) and also performed worse in Boyden-chamber transmigration tests (Fig. 4B). These data were further supported by the observation that MMP9 expression was suppressed by cadaverine treatment (Fig 4C). Metabolic changes evoked by cadaverine administration were assessed using the Seahorse flux analyzer. Cadaverine treatment reduced glycolytic flux (Fig. 4D) that is a characteristic of breast cancer stroma cells [36]. The“sternness” of 4T1 cells was assessed using the aldefluor assay and a reduction in cancer cell sternness was found (Fig. 4E).
The influence of cadaverine administration on the proliferation of cultured breast cancer cell lines was also investigated on five different established breast cancer cell lines of which four were of human (MD -MBA-231, SKBR3, ZR-75-1 and MCF7), while one was of murine origin (4T1). The cadaverine concentration used corre sponded to the reference concentration of cadaverine in human serum (100-800 nM) [34, 35, 36]. Cadaverine slowed proliferation of 4T1, MDA-MB-231 and SKBR-3 cells as measured in SRB assay (Fig. 2A) or in colony forming assays (Fig. 2B). Importantly, the same concentrations of cadaverine did not hinder the proliferation of non-transformed primary human skin fibroblasts (Fig. 2A). We assessed whether slower proliferation could be due to the toxicity of cadaverine to cells. The proportion of the PI positive cells did not increase upon cadaverine treatment (Fig. 2D), nor did the apoptotic fraction in 4T1 cells (Fig. 2C).
Cadaverine exerts its beneficial effects through Trace Amino Acid Receptors (TAARs). The trace amino ac id receptor family serves as receptors for cadaverine [37, 38, 39]. Indeed, higher expression of TAAR1, TAAR2, TAAR4, TAAR5 and TAAR9 provided better survival in breast cancer (Fig 5A). As TAAR receptors are G protein-dependent receptors [38] we assessed their involvement by treating 4T1 cells with NF449, a Gsa- subunit-selective G-protein antagonist, a treatment that abolished the anti-EMT effect of cadaverine (Fig. 5B).
Interestingly, it was found that E. coli FdcC levels in the feces of patients having E-cadherin negative breast cancer are lower than in the feces of patients having E-cadherin positive breast cancer. (Fig. 8) E-cadherin ex pression in breast cancer has been linked to disease progression, metastasis, aggressiveness of the tumor and reduced overall survival [76]. This finding further supports the tumor suppressive role of cadaverine.
Some aspects of the invention are summerized below:
1. A method for diagnosing early stage breast cancer in a human subject is provided, comprising measuring the abundance of at least one bacterium species which is capable of producing cadaverine un der physiological conditions in the human body, in a test sample derived from the subject, wherein the test sam ple comprises human microbiota of said subject, and
wherein a decrease in the abundance of the at least one species in the test sample compared to a reference value of abundance of the at least one bacterium species typical of the absence of early stage breast cancer, is indicative of early stage breast cancer in said subject. In the context of the description, early stage breast cancer refers to stage 0 and stage 1 breast cancer, preferably to stage 0 breast cancer or preferably to stage 1 breast cancer.
2. Preferably, the method is for diagnosing breast cancer stage 0 according to the American Joint Com mittee on Cancer (AJCC) TNM system and the test sample is compared to a reference value typical of the ab sence of stage 0 breast cancer. Preferably, the method is for diagnosing breast cancer stage 1 according to the American Joint Committee on Cancer (AJCC) TNM system and the test sample is compared to a reference value typical of the absence of stage 1 breast cancer.
3. Preferably, the abundance of the bacterium species to be measured is measured by measuring the abundance of the DNA sequence coding for or a gene product of the DNA sequence coding for lysine decar boxylase (LDC). Preferably, the DNA sequence coding for LDC is a DNA sequence in the ldc operon and/or in the cad operon or the gene product is a gene product of a gene of the ldc operon and/or of the cad operon. More preferably, the DNA sequence in the ldc operon is ldcC and/or the DNA sequence in the cad operon is cadA or the gene of the ldc operon is ldcC and/or the gene of the cad operon is cadA. The gene product is preferably RNA or protein. The protein is preferably LDC, LdcC protein or CadA protein (lysine decarboxylase). The RNA is preferably mRNA. Preferably, when stage 0 is to be diagnosed, DNA or RNA is used, and when stage 1 is to be disagnosed, protein is used.
4. The method according to any one of points 1 to 3, wherein the test sample comprising human microbiota of said subject comprises gut microbiota.
5. The method according to any one of points 1 to 4, wherein the at least one bacterium species is select ed from Escherichia, Enterobacter and Hafnia. Preferably, the at least one bacterium species is selected from Escherichia coli, Enterobacter cloacae and Hafnia alvei. In preferred embodiments the bacteria to be measured are Escherichia coli, Enterobacter cloacae and Hafnia alvei.
Preferably, when measuring the abundance of E. cloacae and/or H. alvei, the abundance of a DNA se quence coding for LDC or a gene product of thereof is measured, wherein the DNA sequence codes for LdcC protein; when measuring the abundance of E. coli, the abundance of a DNA sequence coding for LDC or a gene product of thereof is measured, wherein the DNA sequence codes for LdcC protein and/or CadA protein.
The test sample may be serum, plasma, whole blood, breast duct fluid, breast tumor tissue or feces. The sample in which the abundance of bacteria is to be measured is preferably feces. The samples to be compared are corresponding samples, e.g. when the abundance of a bacterium species is measured in a feces sample, the
reference value is derived from feces. The reference value is a value calculated from samples of individuals not having breast cancer, preferably early stage breast cancer, more preferably stage 0 breast cancer. Preferably, the reference value is calculated from samples of healthy individuals.
Preferably the bacterium species capable of producing cadaverine under physiological conditions in the human body is capable of said production in the human gastrointestinal tract.
The invention also provides cadaverine for use in the treatment of breast cancer in a patient. A method for treating breast cancer in a patient, comprising administering an effective dose of cadaverine to the patient, is also provided.
In an embodiment, the treatment decreases the number of metastases in said patient.
In yet other embodiments, said treatment decreases the total mass of the primary tumor(s) and/or the total mass of the metastases.
Preferably, said treatment decreases the infiltration rate of the primary tumor(s) into surrounding tissues. More preferably, infiltration of the primary tumor(s) into surrounding tissues (e.g. blood vessel or lymphatic vessels) is prevented.
In preferred embodiments said treatment reverses endothelial-mesenchymal transition in the breast can cer cells. In preferred embodiments endothelial-mesenchymal transition is indicated and measured by the de creased expression of any one of the following genes in primary tumor tissue or metastasis tissue: MMP2, MMP3, Tgfb3, FgFbpl, Erbb3, Erl, Notchl, IgFbp4, Sppl, Krtl4, DSC2 and combinations thereof. In pre ferred embodiments the expression of any one of the following genes in primary tumor tissue or metastasis tissue is decreased: MMP3, Erl.
Preferably, said patient is a human, more preferably a woman.
In preferred embodiments, cadaverine is administered in a dose providing in the patient a serum concen tration corresponding to the reference physiological serum concentration of cadaverine. Preferably, cadaverine is administered in a dose that provides about 50 nM/1 to 500 mM/l serum concentration in the patient. More preferably, cadaverine is administered in a dose that provides about 100 to 800 nM/1 serum concentration in the patient. In preferred embodiments, cadaverine is administered in a dose of about 50 nmol/kg to 500 mM/kg, preferably about 100 to 800 nM/kg or in a dose of 0.1 - 1000 mg daily.
Preferably, cadaverine is used to treat early stage breast cancer, in particular stage 0 breast cancer. Pref erably, cadaverine is used as an adjuvant therapy, together with a further anti -cancer treatment.
EXAMPLES
Materials and methods
Chemicals
Designed primers, cadaverine and putrescine were from Sigma-Aldrich (St. Louis, MI, USA). Antibodies were from Cell Signaling Technology (Beverly, MA, USA), Abeam (Cambridge, UK) or from Thermo Fisher Scientific (Rockford, USA) unless otherwise stated.
Cell culture
4T1 murine breast cancer cells were maintained in RPMI-1640 (Sigma-Aldrich, R5886) medium containing
10 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine and 1 % pyruvate at 37 °C with 5 % C02.
MDA-MB-231 and SK-BR-3 human breast cancer cells were maintained in DMEM (Sigma- Aldrich, 1000 mg/1 glucose, D5546) containing 10 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine and 10 mM HEPES at 37 °C with 5 % C02.
ZR-75-1 human breast cancer cells were maintained in RPMI-1640 (Sigma- Aldrich, R5886) medium con taining 10 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine at 37 °C with 5 % C02.
MCF-7 human breast cancer cells were maintained in MEM (Sigma-Aldrich, M8042) medium containing 10 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine and 10 mM HEPES at 37 °C with 5 % C02.
Human primary fibroblast cells were maintained in DMEM (Sigma-Aldrich, 1000 mg/1 glucose, D5546) containing 20 % FBS, 1 % penicillin/streptomycin, 2 mM L-glutamine and 10 mM HEPES at 37 °C with 5 % C02.
Sulphorhodamine B assay
Cells were seeded in 96-well plates (4T1- 1500 cells/well; MDA-MB-231 and ZR-75-1 - 3000 cells/well; SK-BR-3, MCF-7 and human fibroblast - 5000 cells/well) and were let to attach overnight. Cells were treated with different concentration of cadaverine (Sigma Aldrich, C8561) for 48 hours. After 2 days cells were fixed by the addition of 50 % trichloroacetic acid (TCA, final concentration: 10 %) and the plate was incubated for 1 hour at 4 °C. The plate was then washed 5 times with water and was stained with 0.4 % (w/v) sulphorhodamine B solution in 1 % acetic acid. Unbound dye was removed by washing 5 times with 1 % acetic acid. Bound stain was solubilized with 10 mM Tris base and the absorbance was measured at 540 nm.
Colony formation assay
Cells were seeded in 6-well plates (4T1- 750 cells/well; MDA-MB-231, SKBR-3, ZR-75-1 and MCF-7- 1000 cells/well) in complete medium and were treated with the indicated concentrations of cadaverine for 4 days. At the end of the treatment plates were washed in PBS. Colonies were fixed in 4 % PFA for 30 minutes, dried and stained with the solution of May-Griinwald-Giemsa for 30 minutes. Plates were washed with water and the colonies, containing at least 50 cells, were counted using Image J software.
Detection of cell death
For the detection of cell death we used simple propidium iodide (PI, Biotium, Fremont, CA, 40016) uptake assays, while to differentiate between apoptosis and necrosis we used an Annexin V+PI double staining assay kit (Invitrogen, Oregon, USA, V13242).
For PI uptake cells were seeded in 6-well plates (MDA-MB-231, ZR-75-1 and MCF-7 - 100,000 cells/well; SKBR-3 and human fibroblast - 200,000 cells/well). After 2 days of cadaverine treatment cells were stained with 100 pg/rnl propidium iodide for 30 min at 37 °C. Supernatant was collected in FACS tubes, cells were washed with PBS and collected in the same FACS tubes (trypsin: PBS 1:1) then samples were analyzed by flow cytometry (FACSCalibur, BD Biosciences).
4T1 cells were seeded in 6-well plates (50,000 cells/well) and treated with the indicated cadaverine concen trations for 2 days. Cells were harvested in FACS tubes, washed once with cold PBS and stained with 100 pg/rnl PI solution and 5 pi FITC Annexin V (Component A) according to the instructions of the apoptosis kit. The number of apoptotic cells was measured with flow cytometry.
Electric Cell-substrate Impedance Sensing (ECIS)
4T1 cells were seeded on type 8W10E arrays (20,000 cells/well) then treated with 0.1 mM cadaverine. ECIS (Electric cell-substrate impedance sensing) model ZQ, Applied BioPhysics Inc. (Troy, NY, USA) was used to monitor transcellular electric resistance of control and cadaverine treated cells for 20 hours before the treatment, and total impedance values were measured for additional 48 hours upon the indicated cadaverine treatment. Multifrequency measurements were taken at 62.5, 125, 250, 500, 1000, 2000, 4000, 8000, 16000, 32000, 64000 Hz. The reference well was set to a no-cell control with complete medium.
Immunocytochemistry
4T1 cells were grown on coverslips, and treated with the indicated concentration of cadaverine for 48 hours. To investigate the effect of TAARs, 5mM NF449 (Bio-Techne R&D Systems Kft, 627034-85-9) G-protein in hibitor was also added to cadaverine-treated cells. Cells were washed with PBS, fixed with 4% PFA for 15 minutes and permeabilized using 1 % Triton X-100 for 5 minutes. After washing twice with PBS, cells were blocked with 1 % BSA for one hour at room temperature. For direct labelling of the actin cytoskeleton, fixed cells were incubated with TexasRed-X Phalloidin (1:150; Thermo Fisher Scientific) for an hour, followed by several washing steps with PBS. For visualizing MMP9 protein in 4T1 cells, mMMP9 primary antibody (1:1000, Abeam) was applied overnight on cells in a humid chamber at 4°C. Subsequently, primary antibody was visualized by a goat anti -rabbit secondary antibody (1:500, Thermo Fisher Scientific). Cell nuclei were visualized with TO-PRO-3 iodide (1:1000, Thermo Fisher Scientific), or DAPI (1:10, Thermo Fisher Scien tific). Coverslips were rinsed and mounted in Mowiol/Dabco solution. Confocal images were acquired with Feica SP8 confocal microscope and FAS AF v3.1.3 software. Intensity was calculated using Image J software. Materials used in immunocytochemistry assays are summarized in Table 1.
Table 1 Fist of antibodies used for Western blot /Immunocytochemistry
Bacterial FdcC and CadA quantitation
The human DNA library was published in Goedert, J.J., et al. Investigation of the association between the fecal microbiota and breast cancer in postmenopausal women: a population -based case-control pilot study. J Natl Cancer Inst. 107, djvl47. (2015) [17]. For the determination of the abundance of lysine decarboxylase in human samples 10 ng of DNA was used for qPCR reactions. Primers are listed in Table 2. The amplicons were subsequently sequenced using the same primers as listed in Table 2.
Table 2 Primers for the determination of the abundance of Cad A and LdcC using RT-qPCR
mRNA isolation and quantitation, EMT screening
Total RNA from cells were prepared using TRIzol reagent (Invitrogen, TR118). 2 pg RNA was reverse transcripted using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA, 4368813) according to the manufacturer’s instructions. qPCRBIO SyGreen Lo-ROX Supermix (PCR
Biosystems Ltd, London, UK, PB20.11 -05) was used for the qPCR reactions, the expression level of the genes was detected with Light-Cycler 480 Detection System (Roche Applied Science). Geometric mean of 36B4 and cyclophyllin or GAPDH was used for normalization. Primers used in RT-qPCR reactions are listed in Table 3.
Table 3. Primers used in the RT-qPCR reactions
EMT genes differentially regulated upon cadaverine treatment are listed in Table4. Table 4. List of EMT genes differentially regulated upon cadaverine treatment
Table 5. Significant changes in gene expression of 4T1 cells and tumor samples
V alidation of E. coli LdcC antibody
DH5a Escherichia coli were seeded in liquid LB medium. Cells were incubated at 37°C overnight with gen- tie shaking. E. coli cells were then collected with centrifugation and proteins were isolated using RIPA buffer
(50 inM Tris, 150 inM NaCl, 0.1 % SDS, 1 % TritonX 100, 0.5 % sodium deoxycholate, 1 mM EDTA, 1 inM Na3V04, 1 mM NaF, 1 mM PMSF, protease inhibitor coctail). Cell wall and membrane were dismpted using ultrasound sonicator (Branson Ultrasonic Sonifier S-250A, Thermo Fisher Scientific) 3 times for 30 seconds at 50 % amplitude. Samples were analyzed by SDS -PAGE followed by Western blotting.
Fecal sample preparation
RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1 % SDS, 1 % TritonX 100, 0.5 % sodium deoxycolate, 1 mM EDTA, 1 mM Na3V04, 1 mM NaF, 1 mM PMSF, protease inhibitor coctail) was used to lyse cells of fecal sam ples. Samples were sonicated (Qsonica Q125 Sonicator, Newtown, Connecticut) 3 times for 30 seconds with 50% amplitude. After centrifugation, 25 mΐ 5 X SDS sample buffer (50 % glycerol, 10 % SDS, 310 mM Tris HC1, pH 6.8, 100 mM DTT, 0.01 % bromophenol blue) and 8 mΐ b-mercaptoethanol were added to every 100 mΐ supernatant. Samples were heated 10 minutes at 96 °C and kept on ice until loading.
SDS-PAGE and Western blotting
Protein extracts were separated on 8% SDS polyacrylamide gels and transferred onto nitrocellulose mem branes by electroblotting. Then membranes were blocked with 5 % BSA, and incubated with anti-Fysine decar boxylase primary antibody (1: 100, Abeam) for overnight at 4 °C. The membranes were washed with IX TBS- TWEEN and incubated with IgG HRP conjugated secondary antibody (1:2000, Cell Signaling Technology).
Bands were visualized by enhanced chemiluminescence reaction (SuperSignal West Pico Solutions, Thermo Fisher Scientific, 34578) using Chemidock Touch system. Densitometry was performed using the Image J soft-
ware. Primary and secondary antibodies, used in this study, are listed in Table 1.
Scratch assay
4T1 cells were plated in 6-well plates (150,000 cells/well) and were grown overnight. The plates were man ually scratched with sterile 200 mΐ pipette tip, followed by washing the cells with complete growth medium. Then cells were treated with 0.1 mM cadaverine in a 37°C thermostat and were monitored every hour for 2 days using JuLi Br Live cell movie analyzer (NanoEnTek Inc., Seoul, Korea).
Invasion
Matrigel invasion assay was carried out on 4T1 cells using Coming BioCoat Matrigel Invasion Chamber (354480). 4T1 cells were seeded in the chambers (50,000 cells/well) in semm free medium, and were grown overnight. Cells were then treated with different concentration of cadaverine (0.1 mM, 0.3 mM, 0.8 mM). The lower chamber contained full 4T1 medium with 100 ng/ml SDFl-alpha (Sigma, SRP4388) as chemoattractant. After 48 hours of cadaverine treatment cells were prepared according to the manufacturer’s instructions and stained with Hematoxylin-Eosin (VWR, 340374T and 341972Q) dye. Cells were then pictured with Opera Phenix High Content Screening System and pictures were analysed using Harmony 4.6 Software. Invasion in dex was calculated from the percentage of invading cells through matrigel membrane and control membrane.
Seahorse flux analysis
4T1 cells were seeded in 96-well Seahorse assay plates (1500 cells/well) and treated with vehicle and cadaverine for 48 hours. Cells were monitored using XF96 oxymeter (Seahorse Biosciences, North Billerica, MA, USA) to measure the changes in oxygen consumption rate (OCR) and in pH (ECAR) after cadaverine treatment similarly to [27]. Data were normalized to protein content.
Determination of lipid peroxidation (TB ARS)
Lipid peroxidation was measured by detemining the production rate of thiobarbituric acid reactive substrate (TBARS). 4T1 cells were seeded in T75 flasks and allowed to adhere overnight. Cells were exposed to cadaverine for 48 hours, then collected by centrifugation. 8.1 % SDS, 20 % acetic acid, 0.8 % thiobarbituric acid (TBA) and distilled water was added to the pellet and was heated at 96°C for 1 hour in thermoblock. Sam ples were cooled on ice and centrifugated, the absorbance of the supernatant was measured at 540 nm. In control and cadaverine-treated (A) MDA-MB-231 (n=l in triplicates) and (B) SKBR-3 (n=l in triplicates) breast cancer cells morphology of the actin cytoskeleton was assessed after Texas Red-X Phallodin+To-Pro-3 staining. Ratio (%) of epithelial and mesenchymal cells was shown on bar charts. Significance was calculated using Chi-square test in Microsoft Excel. No changes in lipid oxidation were detected.
Aldefluor assay
The level of the enzyme aldehyde dehydrogenase (ALDH) was determined on 4T1 cells using cadaverine treatment. Cells were seeded on 6 well plates (50000 cells/well) and treated with different concentration of
cadaverine (0.1 mM, 0.3 mM, 0.8 mM) for 2 days. Cells were then collected and prepared according to the manu facturer’s instructions. We used SKBR-3 cell line for positive control. Changes in the level of ALDH was mesured using flow cytometry and the results were analysed with flowing software 2.5.1.
Animal study
Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Debrecen and the National Board for Animal Experimentation (1/2015/DEMAB) and were carried out ac cording to the NIH guidelines (Guide for the care and use of laboratory animals) and applicable national laws. Animal studies are reported in compliance with the ARRIVE guidelines [72, 73].
We used BALB/c female mice (4 months of age, 20-25g). Animals were bred in the“specific pathogen free” zone of the Animal Facility at the University of Debrecen, and kept in the“minimal disease” zone during the experiments. 4 mice were housed in one cage (standard block shape 365 c 207 c 140 mm, surface 530 cm2; 1284 L Eurostandard Type II. L from Techniplast). Dark/light cycle was 12 h, and temperature was 22 ± 1°C. Mice had ad libitum access to food and water (sterilized tap water). A total of 32 female mice were used in the study, 16 randomly selected control and 16 cadaverine fed mice. The study was performed in two runs at two different occasions, each run comprising of 8 vehicle -treated and 8 cadaverine-treated mice.
Tumor was formed in mice by the grafting of 4T1 cells. 4T1 cells were suspended (2xl06/ml) in ice cold PBS-matrigel (1: 1, Sigma- Aldrich) at 1: 1 ratio. 16 female BALB/c mice received 50 pL injections to the ingui nal fat pads below the lower abdominal nipples on both sides (105 cells/injection site).
Animals received daily oral cadaverine treatment. Cadaverine stock was prepared in sterilized tap water at lOOx concentration (15 mM) and the stock was stored at -20°C. Cadaverine stock was diluted each day to a working concentration of 150 mM in sterile tap water before the treatment. Animals received a daily oral dose of 100 m1/30 g body weight from cadaverine solution (8 mice) or vehicle (sterilized tap water, 8 mice). Researchers administering cadaverine and vehicle solutions were blinded. Treatment was carried out every day during the morning hours between 9am and 11am. Mice were sacrificed on day 14 post grafting.
During autopsy primary tumors were scored based on their infiltration rate into surrounding tissues.“Low infiltration” class means that primer tumor remained in the mammary fat pads without any attachment to muscle tissues. In case the tumor mass attached to the muscle tissue but did not penetrate the abdominal wall, it classi fied as a“medium infiltration” tumor. If the tumor grew into the muscle tissue and totally penetrated the ab dominal wall, it was scored as a“high infiltration” tumor.
Both primary and metastatic tumor masses were removed from mice and were measured on analytical bal ance in pre weighed Eppendorf tubes.
Human studies
We assessed the abundance of the bacterial DNA coding for lysine decarboxylase in human fecal DNA samples. The human fecal samples were collected from healthy women and breast cancer patients by collabora tors at the National Cancer Institute (NCI), Kaiser Permanente Colorado (KPCO), the Institute for Genome Sciences at the University of Maryland School of Medicine, and RTI International. The study protocol and all study materials were approved by the Institutional Review Boards at KPCO, NCI, and RTI International (IRB number 11CN235). The primary study results were published in [17]. Fecal samples from 46 breast cancer pa-
tients (stage 0-3) and 48 control subjects (table 6., cohort 1). All females.
Table 6. Cohort 1
Another cohort was used to assess LdcC protein in the feces of healthy volunteers and breast cancer patients. The collection and biobanking of feces was authorized by the Hungarian national authority (ETT). Patients and healthy volunteers meeting the following criteria were excluded from the study according to the corresponding national guideline for fecal transplantation [34]: 1) has previous history of breast cancer or had been operated due to neoplasia, 2) has a disease of unknown origin, 3) has chronic contagious disease, 4) had contagious diar rhea 6 months prior to enrollment, 5) taken antibiotics in the 6 months prior to enrollment, 6) had chemothera py, biological therapy or immunosuppressive therapy 6 months prior to enrollment, 7) used intravenous drugs 12 months prior to enrollment, 8) had piercing, tattooing, acupuncture or other endangering behavior or action 12 months prior to enrollment, 9) exposition to an allergen to which the enrolled individual had been sensitized to, 10) undervent colonoscopy 12 months prior to enrollment. First morning feces was sampled; samples were frozen and deposited in the biobank two hours after defecation. Samples were stored at -70°C until subsequent use.
E-cadherin expression and E. coli LDcC expression
Fecal protein was prepared from the feces of E-cadherin positive (n=29) and negative (n=5) breast cancer cases. Protein was separated using SDS-PAGE. Proteins were transferred to nitrocellulose membrane and was probed for E. coli LdcC protein. Signal was developed using ECL that was normalized for total protein meas ured by amido black-staining of the membrane. E-cadherin expression was assessed by routine immunohisto- chemistry.
Database screening
The kmplot.com database was used to study the link between gene expression levels and breast cancer sur vival in humans. The association of known mutations with breast cancer was retrieved from www.intogen.org/. The sequence of the CadA and LdcC ORFs were retrieved from the KEGG (www.genome.jp/kegg/) database. We assessed the NCBI GEO Profiles with the term“LDC and breast cancer” in June 2018.
Statistical analysis
We used two tailed Student’s t-test for the comparison of two groups unless stated otherwise. Fold data for human fecal DNA assessment were log2 transformed to achieve normal distribution. For multiple comparisons one-way analysis of variance test (ANOVA) was used followed by Tukey’s honestly significance (HSD) post- hoc test. Data is presented as average ± SEM unless stated otherwise. Texas Red-X Phalloidin-labelled fluores cent pictures were analysed using Cell Profiler 2.0 followed by Advanced Cell Classifier 3.0. Statistical analysis was done using Origin 8.6 software unless stated otherwise.
Sequence Listing Free Text <223 > Escherichia coli LdcC forward primer <223 > Escherichia coli LdcC reverse primer <223> Escherichia coli CadA forward primer <223 > Escherichia coli CadA reverse primer <223 > Enterobacter cloacae LdcC forward primer <223 > Enterobacter cloacae LdcC reverse primer <223 > Hafnia alvei LdcC forward primer <223 > Hafnia alvei LdcC reverse primer <223 > MMP2 Murine forward primer
<223 > MMP2 Murine reverse primer
<223 > MMP3 Murine forward primer
<223 > MMP3 Murine reverse primer
<223 > MMP9 Murine forward primer
<223 > MMP9 Murine reverse primer
<223> Krtl5 Murine forward primer
<223> Krt 15 Murine reverse primer
<223 > Sppl Murine forward primer
<223 > Sppl Murine reverse primer
<223 > FgfBpl Murine forward primer
<223 > FgfBpl Murine reverse primer
<223 > Notch 1 Murine forward primer
<223 > Notch 1 Murine reverse primer
<223> Tgfb3 Murine forward primer
<223> Tgfb3 Murine reverse primer
<223 > Erbb3 Murine forward primer
<223 > Erbb3 Murine reverse primer
<223> Erl Murine forward primer
<223> Erl Murine reverse primer
<223 > IgfBp4 Murine forward primer
<223 > IgfBp4 Murine reverse primer
<223> 36B4 Murine forward primer
<223> 36B4 Murine reverse primer
<223 > CyclophillinA Murin forward primer <223 > CyclophillinA Murin reverse primer <223 > GAPDH Murine forward primer <223 > GAPDH Murine reverse primer
<223> T A AR7d Murine forward primer
<223> T A AR7d Murine reverse primer
<223> TAAR8b Murine forward primer
<223> TAAR8b Murine reverse primer
SEQUENCE LISTING
<110> Debreceni Egyetem
<120> Treatment and diagnosis of breast cancer
<130> 124391/KOH
<160> 40
<170> Patentln version 3.5
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Escherichia coli LdcC forward primer
<400> 1
cggcccttat aacctgctgt ttc 23 <210> 2
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Escherichia coli LdcC reverse primer
<400> 2
ccttgtgcca gatcctgaat acg 23 <210> 3
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Escherichia coli CadA forward primer
<400> 3
gtctgtgcgg cgttattttt gac
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Escherichia coli CadA reverse primer
<400> 4
cacccagcgc atattcaaag aag 23 <210> 5
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Enterobacter cloacae LdcC forward primer
<400> 5
atatgatctg aacctgcggg tga 23 <210> 6
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Enterobacter cloacae LdcC reverse primer
<400> 6
aggttctcca gctcaacggt ttc 23 <210> 7
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Hafnia alvei LdcC forward primer
<400> 7
ggtgaactgg gttctctgct tga 23 <210> 8
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Hafnia alvei LdcC reverse primer
<400> 8
agcgggtgct gagtacatac caa
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> MMP2 Murine forward primer
<400> 9
tgggggagat tctcactttg 20
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> MMP2 Murine reverse primer
<400> 10
catcactggg accagtgtct 20
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> MMP3 Murine forward primer
<400> 11
tgggactcta ccactcagcc aag 23
<210> 12
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> MMP3 Murine reverse primer
<400> 12
tgcacattgg tgatgtctca ggt 23
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> MMP9 Murine forward primer
<400> 13
cattcgcgtg gataaggagt 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> MMP9 Murine reverse primer
<400> 14
acctggttca cctcatggtc 20
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Krtl5 Murine forward primer
<400> 15
gaagaggcca acactgaact gga 23
<210> 16
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Krtl4 Murine reverse primer
<400> 16
aggctctgct ccgtctcaaa ctt 23
<210> 17
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Sppl Murine forward primer
<400> 17
gattggcagt gatttgcttt tgc 23
<210> 18
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Sppl Murine reverse primer
<400> 18
ttctgcttct gagatgggtc agg 23
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> FgfBpl Murine forward primer
<400> 19
caaggtccaa gaagctgtct cca 23
<210> 20
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> FgfBpl Murine reverse primer
<400> 20
agctccaaga ttccccacag aac 23
<210> 21
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Notchl Murine forward primer
<400> 21
ccttcacctg tctgtgtcca cct 23
<210> 22
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Notchl Murine reverse primer
<400> 22
tcacagtggt actgcgtgtt ggt 23
<210> 23
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Tgfb3 Murine forward primer
<400> 23
ggcgtctcaa gaagcaaaag gat 23
<210> 24
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Tgfb3 Murine reverse primer
<400> 24
ccttaggttc gtggacccat ttc 23
<210> 25
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Erbb3 Murine forward primer
<400> 25
tacttgcctc tgggctctct cct 23
<210> 26
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Erbb3 Murine reverse primer
<400> 26
cacctggact tgactcggtg act 23
<210> 27
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Erl Murine forward primer
<400> 27
gaccatgacc cttcacacca aag 23
<210> 28
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Erl Murine reverse primer
<400> 28
ctcggggtag ttgaacacag tgg 23
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> IgfBp4 Murine forward primer
<400> 29
caagatgaag atcgtgggga cac 23
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> IgfBp4 Murine reverse primer
<400> 30
cagtttggaa tggggatgat gaa 23
<210> 31
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> 36B4 Murine forward primer
<400> 31
agattcggga tatgctgttg g 21
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> 36B4 Murine reverse primer
<400> 32
aaagcctgga agaaggaggt c 21
<210> 33
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> CyclophillinA Murin forward primer
<400> 33
tggagagcac caagacagac a 21
<210> 34
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> CyclophillinA Murine reverse primer
<400> 34
tgccggagtc gacaatgat 19
<210> 35
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> GAPDH Murine forward primer
<400> 35
caaggtcatc catgacaact ttg 23
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> GAPDH Murine reverse primer
<400> 36
ggccatccac agtcttctgg 20
<210> 37
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> TAAR7d Murine forward primer
<400> 37
attgatgcct tccttgggtt cat 23 <210> 38
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> TAAR7d Murine reverse primer
<400> 38
caggaaacag gttggtggtt gag 23 <210> 39
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> TAAR8b Murine forward primer
<400> 39
actcttctgc cctccacctg tgc 23 <210> 40
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> TAAR8b Murine reverse primer
<400> 40
ttgacaacga tttggcagcc ccc 23
REFERENCES
1. Macfabe, D. Autism: metabolism, mitochondria, and the microbiome. Glob Adv Health Med 2, 52-66 (2013).
2. Mamvada, P., Leone, V., Kaplan, L.M. & Chang, E.B. The Human Microbiome and Obesity: Moving be yond Associations. Cell Host Microbe. 22, 589-599. (2017).
3. Fulbright, L.E., Ellermann, M. & Arthur, J.C. The microbiome and the hallmarks of cancer. PLoS Pathog. 13, el006480. (2017).
4. Kundu, P., Blacher, E., Elinav, E. & Pettersson, S. Our Gut Microbiome: The Evolving Inner Self. Cell. 171, 1481-1493. doi: 1410.1016/j.cell.2017.1411.1024. (2017).
5. Zitvogel, L., Ayyoub, M., Routy, B. & Kroemer, G. Microbiome and Anticancer Immunosurveillance. Cell.
165, 276-287. doi: 210.1016/j.cell.2016.1003.1001. (2016).
6. Schwabe, R.F. & Jobin, C. The microbiome and cancer. Nat Rev Cancer 13, 800-812 (2013).
7. Plottel, C.S. & Blaser, M.J. Microbiome and malignancy. Cell Host Microbe 10, 324-335 (2011).
8. Garrett, W.S. Cancer and the microbiota. Science. 348, 80-86. (2015).
9. Yu, H., et al. Urinary microbiota in patients with prostate cancer and benign prostatic hyperplasia. Archives of medical science : AMS 11, 385-394 (2015).
10. Chase, D., Goulder, A., Zenhausern, F., Monk, B. & Herbst-Kralovetz, M. The vaginal and gastrointestinal microbiomes in gynecologic cancers: a review of applications in etiology, symptoms and treatment. Gynecol Oncol 138, 190-200 (2015).
11. Yu, Y., Champer, J., Beynet, D., Kim, J. & Friedman, A.J. The role of the cutaneous microbiome in skin cancer: lessons learned from the gut. Journal of drugs in dermatology : JDD 14, 461-465 (2015).
12. Gui, Q.F., Lu, H.F., Zhang, C.X., Xu, Z.R. & Yang, Y.H. Well-balanced commensal microbiota contributes to anti-cancer response in a lung cancer mouse model. Genetics and molecular research : GMR 14, 5642- 5651 (2015).
13. Garrett, W.S. Cancer and the microbiota. Science 348, 80-86 (2015).
14. Yamamoto, M.L. & Schiestl, R.H. Lymphoma caused by intestinal microbiota. International journal of envi ronmental research and public health 11, 9038-9049 (2014).
15. Yamamoto, M.L. & Schiestl, R.H. Intestinal microbiome and lymphoma development. Cancer J. 20, 190- 194. (2014).
16. Goedert, J.J., et al. Postmenopausal breast cancer and oestrogen associations with the IgA-coated and IgA- noncoated faecal microbiota. Br J Cancer 23, 435 (2018).
17. Goedert, J.J., et al. Investigation of the association between the fecal microbiota and breast cancer in post menopausal women: a population-based case-control pilot study. J Natl Cancer Inst. 107, djvl47. (2015).
18. Fuhrman, B.J., et al. Associations of the fecal microbiome with urinary estrogens and estrogen metabolites in postmenopausal women. J Clin Endocrinol Metab. 99, 4632-4640. (2014).
19. Flores, R., et al. Fecal microbial determinants of fecal and systemic estrogens and estrogen metabolites: a cross-sectional study. J Transl Med. 10, 253 (2012).
20. Xuan, C., et al. Microbial dysbiosis is associated with human breast cancer. PLoS One. 9, e83744. (2014).
21. Hieken, T.J., et al. The Microbiome of Aseptically Collected Human Breast Tissue in Benign and Malignant Disease. Sci Rep. 6, 30751. (2016).
22. Chan, A. A., et al. Characterization of the microbiome of nipple aspirate fluid of breast cancer survivors. Sci Rep. 6, 28061 (2016).
23. Urbaniak, C., et al. The Microbiota of Breast Tissue and Its Association with Breast Cancer. Appl Environ Microbiol. 82, 5039-5048. (2016).
24. Yoshimoto, S., et al. Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature. 499, 97-101. (2013).
25. Xie, G., et al. Dysregulated hepatic bile acids collaboratively promote liver carcinogenesis. Int J Cancer. 139, 1764-1775. (2016).
26. Luu, T.H., et al. Lithocholic bile acid inhibits lipogenesis and induces apoptosis in breast cancer cells. Cell Oncol (Dordr). 41, 13-24. (2018).
27. Miko, E., et al. Lithocholic acid, a bacterial metabolite reduces breast cancer cell proliferation and aggres siveness. Biochim Biophys Acta, doi: 10.1016/j.bbabio.2018.1004.1002 (2018).
28. Rowland, I.R. Role of the gut flora in toxicity and cancer, (Academic Press, Carshalton, UK, 1988).
29. Shellman, Z., et al. Bile acids: a potential role in the pathogenesis of pharyngeal malignancy. Clin Otolaryngol. 42, 969-973. (2017).
30. Dapito, D.H., et al. Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4. Cancer Cell 21, 504-516 (2012).
31. Bindels, L.B., et al. Gut microbiota-derived propionate reduces cancer cell proliferation in the liver. Br J Cancer 107, 1337-1344 (2012).
32. Miller-Fleming, L., Olin-Sandoval, V., Campbell, K. & Raiser, M. Remaining Mysteries of Molecular Biolo gy: The Role of Polyamines in the Cell. J Mol Biol. 427, 3389-3406. (2015).
33. Seiler, N. Catabolism of polyamines. Amino Acids. 26, 217-233. (2004).
34. Loser, C., Folsch, U.R., Paprotny, C. & Creutzfeldt, W. Polyamine concentrations in pancreatic tissue, serum, and urine of patients with pancreatic cancer. Pancreas. 5, 119-127. (1990).
35. Loser, C., Folsch, U.R., Paprotny, C. & Creutzfeldt, W. Polyamines in colorectal cancer. Evaluation of poly amine concentrations in the colon tissue, serum, and urine of 50 patients with colorectal cancer. Cancer. 65, 958-966. (1990).
36. Pavlides, S., et al. The reverse Warburg effect: aerobic glycolysis in cancer associated fibroblasts and the tumor stroma. Cell Cycle 8, 3984-4001 (2009).
37. Liberies, S.D. Trace amine-associated receptors: ligands, neural circuits, and behaviors. Curr Opin Neurobiol.
34, 1-7 (2015).
38. Vattai, A., et al. Increased trace amine-associated receptor 1 (TAAR1) expression is associated with a posi tive survival rate in patients with breast cancer. J Cancer Res Clin Oncol 13, 017-2420 (2017).
39. NCBI_GEO_Profiles. LDC expression in control, DCIS, ICS. Vol. 2018 (https://www.ncbi. nlm.nih.gov/geo/tools/profileGraph.cgi?ID=GDS3853:201744_s_at, 2018).
40. NCBI_GEO_Profiles. LDC expression in epithelium and stroma of normal breast and invasive breast cancer.
Vol. 2018 (https://www.ncbi. nlm.nih.gov/geo/tools/profileGraph.cgi?ID=GDS3324:201744_s_at, 2018).
41. NCBI_GEO_Profiles. LDC expression in ER- and ER+ breast cancer patients, prophylactic mastectomy patients, and normal breast epithelia from reduction mammoplasty patients., Vol. 2018 (https://www.ncbi. nlm.nih.gov/geo/tools/profileGraph.cgi?ID=GDS3716:201744_s_at, 2018).
42. NCBI_GEO_Profiles. Normal epithelium vs. breast cancer epithelium in patients.
(https://www.ncbi. nlm.nih.gov/geo/tools/profileGraph. cgi?ID=GDS3139:201744_s_at, 2018).
43. NCBI_GEO_Profiles. LDC expression in control vs. non-basal vs. basal-like breast cancer. Vol. 2018 (https://www.ncbi. nlm.nih.gov/geo/tools/profileGraph.cgi?ID=GDS2250:201744_s_at, 2018).
44. Mashige, F., et al. Clinical usefulness of an enzymatic determination of total urinary polyamines, excluding cadaverine. Clin Chem. 34, 2271-2274. (1988).
45. Elitsur, Y., Moshier, J.A., Murthy, R., Barbish, A. & Luk, G.D. Polyamine levels, ornithine decarboxylase (ODC) activity, and ODC-mRNA expression in normal and cancerous human colonocytes. Life Sci 50, 1417-
1424. (1992).
46. Khuhawar, M.Y. & Qureshi, G.A. Polyamines as cancer markers: applicable separation methods. J Chromatogr B Biomed Sci Appl. 764, 385-407. (2001).
47. Liu, R., et al. Determination of polyamine metabolome in plasma and urine by ultrahigh performance liquid chromatography-tandem mass spectrometry method: application to identify potential markers for human he patic cancer. Anal Chim Acta. 791, 36-45. (2013).
48. Olaya, J., Neopikhanov, V. & Uribe, A. Lipopolysaccharide of Escherichia coli, polyamines, and acetic acid stimulate cell proliferation in intestinal epithelial cells. In Vitro Cell Dev Biol Anim. 35, 43-48. (1999).
49. Aizencang, G., et al. Antiproliferative effects of Nl,N4-dibenzylputrescine in human and rodent tumor cells.
Cell Mol Biol (Noisy-le-grand). 44, 615-625. (1998).
50. Velicer, C.M., et al. Antibiotic use in relation to the risk of breast cancer. JAMA. 291, 827-835. (2004).
51. Velicer, C.M., Heckbert, S.R., Rutter, C., Lampe, J.W. & Malone, K. Association between antibiotic use prior to breast cancer diagnosis and breast tumour characteristics (United States). Cancer Causes Control. 17, 307-313. (2006).
52. Friedman, G.D., et al. Antibiotics and risk of breast cancer: up to 9 years of follow-up of 2.1 million women.
Cancer Epidemiol Biomarkers Prev. 15, 2102-2106. (2006).
53. Wirtz, H.S., et al. Frequent antibiotic use and second breast cancer events. Cancer Epidemiol Biomarkers Prev. 22, 1588-1599. (2013).
54. Tamim, H.M., Hanley, J.A., Hajeer, A.H., Boivin, J.F. & Collet, J.P. Risk of breast cancer in relation to anti biotic use. Pharmacoepidemiol Drug Saf. 17, 144-150. (2008).
55. Satram-Hoang, S., et al. A pilot study of male breast cancer in the Veterans Affairs healthcare system. J Envi ron Pathol Toxicol Oncol 29, 235-244. (2010).
56. Fodor, T., et al. Combined Treatment of MCF-7 Cells with AICAR and Methotrexate, Arrests Cell Cycle and Reverses Warburg Metabolism through AMP -Activated Protein Kinase (AMPK) and FOXOl . PLoS One. 11, e0150232. (2016).
57. Nagy, L., et al. Glycogen phosphorylase inhibition improves beta cell function. Br J Pharmacol. 175, 301 - 319. (2018).
58. Szanto, M., et al. Deletion of PARP-2 induces hepatic cholesterol accumulation and decrease in HDL levels.
Biochem Biophys Acta - Molecular Basis of Disease 1842, 594-602 (2014).
59. Mabley, J.G., et al. Suppression of intestinal polyposis in Apcmin/+ mice by targeting the nitric oxide or poly(ADP-ribose) pathways. Mutat.Res. 548, 107-116 (2004).
60. Fiorillo, M., et al. Bergamot natural products eradicate cancer stem cells (CSCs) by targeting mevalonate, Rho-GDI-signalling and mitochondrial metabolism. Biochim Biophys Acta 4, 30061-30066 (2018).
61. Nagy, G.G., Varvolgyi, C., Balogh, Z., Orosi, P. & Paragh, G. [Detailed methodological recommendations for the treatment of Clostridium difficile-associated diarrhea with faecal transplantation]. Orv Hetil. 154, 10- 19. (2013).
62. Depierre D, Jung A, Culebras J, Roth M. Polyamine excretion in the urine of cancer patients. J Clin Chem Clin Biochem. 1983 Jan;21(l):35-7
63. Liang et al Fecal Bacteria Act as Novel Biomarkers for Noninvasive Diagnosis of Colorectal Can- cer.ClinCancer Res. 2017 Apr 15;23(8):2061-2070
64. Wong SH, et al. Quantitation of faecal Fusobacterium improves faecal immunochemical test in detecting advanced colorectal neoplasia Gut 2017;66: 1441-1448
65. Nabizadeh et al. Association of altered gut microbiota composition with chronic urticaria Ann Allergy Asthma Immunol 119 (2017) 48-53
66. Goedert et al. Fecal Microbiota Characteristics of Patients with Colorectal Adenoma Detected by Screen ing: A Population-based Study. EBioMedicine. 2015 Jun; 2(6): 597-603
67. Xie et al. Fecal Clostridium symbiosum for Noninvasive Detection of Early and Advanced Colorectal Can cer: Test and Validation Studies. EBioMedicine. 2017 Nov; 25: 32-40
68. Sommer Advancing gut microbiome research using cultivation Current Opinion in Microbiology 2015, 27: 127-132
69. Lagier et al. Culture of previously uncultured members of the human gut microbiota by culturomics NATURE MICROBIOLOGY DOI: 10.1038/NMICROBIOL.2016.203
70. Browne et al Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation Nature 533 26 May 2016
71. Turnbaugh et al. The human microbiome project: exploring the microbial part of ourselves in a changing world. Nature 2007; 449(7164): 804-810
72. Kilkenny, C, Browne, W.J., Cuthill, I.C., Emerson, M. & Altman, D.G. (2010). Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 8: el000412 doi: 10.1371/journal.pbio.1000412
73. McGrath, J. C., & Lilley, E. (2015). Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. British Journal of Pharmacology, 172(13), 3189-3193. http://doi.org/10.1111/bph.12955
74. Yoshihiro Yamamotol, Yoshihiro Miwa2, Keiko Miyoshi2,Jun-ichi Furuyamal, and Haruo Ohmori (1997)
The Escherichia coli ldcC gene encodes another lysinedecarboxylase, probably a constitutive enzyme. Genes Genet. Syst. (1997) 72, p. 167-172.
75. Inoue, H., Kikuchi, K., and Nishino. M. (1986) Effects of epidermalgrowth factor on the synthesis of DNA and polyamine in iso-proterenol-stimulated murine parotid gland. J. Biochem. 100,605-613.
76. Liu et al. (2016) CDH1 promoter methylation correlates with decreased gene expression and poor prognosis in patients with breast cancer. Oncol. Lett. 11(4) 2635-2643.
Claims
1. A method for determining whether a subject
i) has early stage breast cancer or
ii) has an increased probability of having early stage breast cancer or
iii) is at an increased risk of developing breast cancer,
the method comprising
assessing, in a test sample from the subject, the level of bacterial cadaverine synthesis, the sample comprising microbiota from the subject or
measuring, in a test sample from the subject, the level of cadaverine
wherein
a decreased level of bacterial cadaverine synthesis or
a decreased level of cadaverine, respectively,
indicates that the subject has early stage breast cancer or an increased probability of having early stage breast cancer or is at an increased risk of developing breast cancer.
2. The method according to claim 1
wherein the level of bacterial cadaverine synthesis is assessed by measuring
the abundance of a bacterium species comprising a DNA sequence coding for a lysine decarboxylase (LDC) or
the level of a DNA sequence coding for an LDC of a bacterium species, or
the level of a gene product of a DNA sequence coding for an LDC produced by a bacterium species, wherein
a lower abundance of the bacterium species in the test sample or
a lower level of the DNA sequence coding for an LDC of a bacterium species, or
a lower level of the gene product of a DNA sequence coding for an LDC produced by a bacterium species, respectively,
compared to the corresponding reference value
indicates that the subject has early stage breast cancer or an increased probability of having early stage breast or is at an increased risk of developing breast cancer.
3. The method according to claim 1 or 2, wherein the breast cancer is stage 0 or 1 breast cancer according to the American Joint Committee on Cancer (AJCC) TNM system.
4. The method according to any one of the preceding claims, wherein the DNA sequence coding for LDC is ldcC or cadA.
5. The method according to any one of the preceding claims, wherein the test sample is a feces sample.
6. The method according to any one of the preceding claims, wherein the bacterium species is selected from Escherichia, Enterobacter and Hafnia.
7. The method according to claim 6, wherein the bacterium species is selected from Escherichia coli,
Enterobacter cloacae and Hafnia alvei.
8. The method according to any one of the preceding claims, wherein
a lower abundance of the bacterium species in the test sample or
a lower level of the DNA sequence coding for an LDC of a bacterium species
compared to the corresponding reference value
indicates that the subject has stage 0 breast cancer or an increased probability of having stage 0 breast cancer or the subject is at an increased risk of developing breast cancer.
9. The method according to any one of claims 1 to 7, wherein
a lower level of the gene product of a DNA sequence coding for an LDC produced by a bacterium species compared to a reference sample
indicates that the subject has stage 1 breast cancer or an increased probability of having stage 1 breast cancer.
10. The method according to claim 9, wherein the gene product is LDC protein.
11. The method according to any one of the preceding claims, wherein the subject is a postmenopausal woman.
12. Cadaverine for use in the treatment or prevention of breast cancer in a subject.
13. Cadaverine for use in mitigating cancer initiation and/or promotion and/or progression in a subject.
14. Cadaverine for use according to claim 12 or 13, wherein the subject
has early stage breast cancer or
has an increased probability of having early stage breast cancer or
is at an increased risk of developing breast cancer.
15. Cadaverine for use according to any one of claims 12 to 14, wherein prior to said use
the abundance of a bacterium species comprising a DNA sequence coding for LDC or
the level of a DNA sequence coding for an LDC of a bacterium species or
the level of a gene product of a DNA sequence coding for an LDC produced by a bacterium species is lower in a test sample from the subject comprising microbiota from said subject than the corresponding refer ence value, or
the level of cadaverine in a test sample from the subject is lower than the corresponding reference value.
16. Cadaverine for use according to any one of claims 11 to 15, for use in
decreasing the infiltration rate of primary tumor(s) into surrounding tissues and/or
decreasing the number of metastases and/or
decreasing the total mass of the primary tumor(s) and/or
decreasing the total mass of the metastases.
17. Cadaverine for use according to any one of claims 12 to 16, wherein cadaverine is administered to pro vide or restore physiological serum concentration of cadaverine in said subject.
18. A method for mitigating breast cancer initiation and/or promotion and/or progression in a subject, com prising
measuring in a test sample from said subject, wherein the test sample comprises microbiota from said subject, the abundance of a bacterium species comprising a DNA sequence coding for a lysine decarboxylase (LDC), the level of a DNA sequence coding for an LDC of a bacterium species, or
the level of a gene product of a DNA sequence coding for an LDC produced by a bacterium species, or measuring, in a test sample from said subject, the level of cadaverine,
administering cadaverine to the subject to provide or restore physiological serum concentration of cadaverine if the abundance of the bacterium species comprising a DNA sequence coding for LDC or
the level of the DNA sequence coding for an LDC of a bacterium species or
the level of the gene product of a DNA sequence coding for an LDC produced by a bacterium species or the level of cadaverine, respectively,
is lower than the corresponding reference value, and
monitoring the presence of breast cancer in the subject.
19. A pharmaceutical composition comprising cadaverine and at least one pharmaceutically acceptable ex cipient or carrier.
20. The pharmaceutical composition according to claim 19 for use in mitigating breast cancer initiation and/or promotion and/or progression.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| HUP1800271 | 2018-07-31 | ||
| PCT/HU2019/050038 WO2020025989A1 (en) | 2018-07-31 | 2019-07-31 | Treatment and diagnosis of breast cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP3830300A1 true EP3830300A1 (en) | 2021-06-09 |
Family
ID=89720057
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP19773488.2A Pending EP3830300A1 (en) | 2018-07-31 | 2019-07-31 | Treatment and diagnosis of breast cancer |
Country Status (2)
| Country | Link |
|---|---|
| EP (1) | EP3830300A1 (en) |
| WO (1) | WO2020025989A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022034349A1 (en) * | 2020-08-11 | 2022-02-17 | Debreceni Egyetem | Treatment and diagnosis of breast cancer |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2980709B1 (en) | 2011-09-29 | 2013-10-25 | Nutrialys Medical Nutrition Sa | COMPOSITIONS CONTAINING SPERMINE AND PHARMACEUTICAL COMPOSITIONS CONTAINING SAME |
| KR101390589B1 (en) * | 2012-05-30 | 2014-04-30 | 가천대학교 산학협력단 | Composition for preventing or treating diabetes comprising diamines |
| CA2901737A1 (en) * | 2013-02-19 | 2014-08-28 | John Wayne Cancer Institute | Methods of diagnosing and treating cancer by detecting and manipulating microbes in tumors |
| CN105765383B (en) | 2013-10-28 | 2018-04-10 | 唾液科技有限公司 | Cancer of pancreas is with saliva biomarker and has used diagnostic method of the cancer of pancreas with the cancer of pancreas of saliva biomarker |
| JP6614435B2 (en) | 2015-07-14 | 2019-12-04 | 静岡県公立大学法人 | Breast cancer test method |
-
2019
- 2019-07-31 EP EP19773488.2A patent/EP3830300A1/en active Pending
- 2019-07-31 WO PCT/HU2019/050038 patent/WO2020025989A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO2020025989A1 (en) | 2020-02-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mikó et al. | Lithocholic acid, a bacterial metabolite reduces breast cancer cell proliferation and aggressiveness | |
| Kovács et al. | Cadaverine, a metabolite of the microbiome, reduces breast cancer aggressiveness through trace amino acid receptors | |
| Genua et al. | The role of gut barrier dysfunction and microbiome dysbiosis in colorectal cancer development | |
| Zhang et al. | Vitamin D receptor protects against dysbiosis and tumorigenesis via the JAK/STAT pathway in intestine | |
| Zhong et al. | Gut dysbiosis promotes prostate cancer progression and docetaxel resistance via activating NF-κB-IL6-STAT3 axis | |
| Zhang et al. | Lack of vitamin D receptor leads to hyperfunction of claudin-2 in intestinal inflammatory responses | |
| Li et al. | Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway | |
| Choe et al. | The RUNX2 transcription factor negatively regulates SIRT6 expression to alter glucose metabolism in breast cancer cells | |
| Zhang et al. | The autophagic-lysosomal and ubiquitin proteasome systems are simultaneously activated in the skeletal muscle of gastric cancer patients with cachexia | |
| Xu et al. | Disulfiram/copper markedly induced myeloma cell apoptosis through activation of JNK and intrinsic and extrinsic apoptosis pathways | |
| Palou et al. | Protein expression patterns of ezrin are predictors of progression in T1G3 bladder tumours treated with nonmaintenance bacillus Calmette-Guerin | |
| Zhou et al. | CCL19 suppresses gastric cancer cell proliferation, migration, and invasion through the CCL19/CCR7/AIM2 pathway | |
| Wu et al. | Characterization of site-specific glycosylation of secreted proteins associated with multi-drug resistance of gastric cancer | |
| Rahman et al. | Associations of total arsenic in drinking water, hair and nails with serum vascular endothelial growth factor in arsenic-endemic individuals in Bangladesh | |
| Li et al. | Icariin enhances youth-like features by attenuating the declined gut microbiota in the aged mice | |
| Tabassam et al. | Helicobacter pylori‐associated regulation of forkhead transcription factors FoxO1/3a in human gastric cells | |
| JP2024045362A (en) | Markers for predicting and/or determining therapeutic efficacy of pd-1 signal inhibitor-containing drugs | |
| Sanmamed et al. | Relevance of MIA and S100 serum tumor markers to monitor BRAF inhibitor therapy in metastatic melanoma patients | |
| WO2018229519A1 (en) | Methods for diagnosing breast cancer | |
| Sári et al. | Fecal expression of Escherichia coli lysine decarboxylase (LdcC) is downregulated in E-cadherin negative lobular breast carcinoma | |
| Shibamoto et al. | Vitamin D deficiency exacerbates alcohol-related liver injury via gut barrier disruption and hepatic overload of endotoxin | |
| Li et al. | Potential risk of tamoxifen: gut microbiota and inflammation in mice with breast cancer | |
| EP3830300A1 (en) | Treatment and diagnosis of breast cancer | |
| Xu et al. | Akkermansia muciniphila-derived pentadecanoic acid enhances oxaliplatin sensitivity in gastric cancer by modulating glycolysis | |
| Volpato et al. | Changes in plasma chemokine CC motif ligand 2 levels during treatment with eicosapentaenoic acid predict outcome in patients undergoing surgery for colorectal cancer liver metastasis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: UNKNOWN |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
| 17P | Request for examination filed |
Effective date: 20210226 |
|
| AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
| DAV | Request for validation of the european patent (deleted) | ||
| DAX | Request for extension of the european patent (deleted) | ||
| GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: GRANT OF PATENT IS INTENDED |
|
| INTG | Intention to grant announced |
Effective date: 20240709 |
|
| RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: SEBO, EVA Inventor name: TOTH, JUDIT Inventor name: GOEDERT, JAMES J. Inventor name: VIDA, ANDRAS Inventor name: MIKO, EDIT Inventor name: KOVACS, TUENDE Inventor name: BAY, PETER |