WO2021204109A1 - Utilisation de la p70 s6 kinase nucléaire pour le diagnostic, le pronostic et le traitement du cancer - Google Patents

Utilisation de la p70 s6 kinase nucléaire pour le diagnostic, le pronostic et le traitement du cancer Download PDF

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WO2021204109A1
WO2021204109A1 PCT/CN2021/085641 CN2021085641W WO2021204109A1 WO 2021204109 A1 WO2021204109 A1 WO 2021204109A1 CN 2021085641 W CN2021085641 W CN 2021085641W WO 2021204109 A1 WO2021204109 A1 WO 2021204109A1
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cancer
nuclear
p70s6k
hnrnpa2
metastasis
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Sze Tsai Alice WONG
Kit Yan Sally TO
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The University Of Hong Kong
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • Ovarian cancer is a leading cause of death from gynecological malignancy (Chandra A et al., 2019) . These deaths primarily result from metastatic disease with the cancer cells detached from the primary tumor and spread beyond the ovary and throughout the peritoneal cavity. This peritoneal dissemination is also particularly critical in addressing the challenge of treating ovarian cancer in which current therapies are largely ineffective (5-year survival ⁇ 25%) (Coscia F et al., 2018) . Unraveling the molecular mechanisms underlying the regulation of this process will conceivably help to improve treatment for this deadly disease in the future.
  • EMT Epithelial-to-mesenchymal transition
  • p70 S6 kinase is a downstream effector of the phosphatidylinositol 3-kinase (PI3K) /Akt/mammalian target of rapamycin (mTOR) pathway.
  • PI3K phosphatidylinositol 3-kinase
  • mTOR rapamycin
  • the ribosomal protein S6 is a key component of the translation machinery in the cytoplasm which has a central role in the regulation of cell growth in response to growth factors, cellular energy and nutrient levels.
  • p70 S6K shows predominant cytoplasmic localization in many studies, emerging evidence suggested p70 S6K could translocate into the nucleus in response to various growth factor stimuli (Kim SJ and Kahn CR, 1997; Valovka T et al., 2003; Rosner and Hengstschlager 2011) .
  • p70 S6K plays a pivotal role in other aspects of tumor progression such as metastasis.
  • this was associated with the nuclear localization of p70 S6K .
  • Growth factor stimuli led to p70 S6K phosphorylation and triggered nuclear translocation of p70 S6K .
  • the phosphorylation was sensitive to p70 S6K inhibitors.
  • a method of diagnosing cancer metastasis comprising: (i) measuring phosphorylation of p70 S6K ; or (ii) measuring the binding of p70 S6K to hnRNPA2/B1 in the nucleus, wherein an increased phosphorylation of p70 S6K or increased binding of p70 S6K to hnRNAP2/B1 as compared to a control indicates cancer metastasis.
  • the binding of p70 S6K to hnRNAP2/B1 in the nucleus is detected by immunohistochemistry or subcellular fractionation followed by Western blot analysis.
  • the cancer is ovarian cancer, breast cancer, colon cancer and liver cancer.
  • the cancer is peritoneal metastasis or blood-borne metastasis.
  • a method of early detection of peritoneal dissemination in ovarian tumor comprising: (i) measuring phosphorylation of p70 S6K ; or (ii) measuring the binding of p70 S6K to hnRNPA2/B1 in the nucleus, wherein an increased phosphorylation of p70 S6K or increased binding of p70 S6K to hnRNAP2/B1 as compared to a control indicates cancer metastasis.
  • the binding of p70 S6K to hnRNAP2/B1 in the nucleus is detected by immunohistochemistry or subcellular fractionation followed by Western blot analysis.
  • the ovarian cancer is peritoneal metastasis or blood-borne metastasis.
  • a method of cancer treatment comprising: (i) selective inhibition of nuclear p70 S6K activity; (ii) selective inhibition of hnRNPA2/B1; or (iii) prevention of binding of p70 S6K to hnRNPA2/B1 in the nucleus.
  • the cancer is ovarian cancer, breast cancer, colon cancer and liver cancer.
  • the cancer is metastatic.
  • the cancer is peritoneal metastasis or blood-borne metastasis.
  • the hnRNPA2/B1 is inhibited by sRNA.
  • the cancer treatment inhibits epithelial-to-mesenchymal transition.
  • the cancer treatment regulates alternative splicing in cancer.
  • Fig. 1 Nuclear localization of p70 S6K is positively correlated with the metastatic propensities and promotes EMT of human ovarian cancer cells.
  • Western blotting analysis of cytosolic and nuclear fractions of (A) ascites tumor cells from patients with different stage ovarian carcinoma; (B) two isogenic cell line pairs which are separated from HEYA8 and SKOV3. ip1 according to their different metastatic potentials (HM: high-metastatic; NM: non-metastatic) ; (C) multiple human ovarian cancer cell lines with different epithelial and mesenchymal states.
  • C cytosol
  • N nuclei.
  • p70 S6K re-localizes to the nucleus in a phosphorylation-dependent manner.
  • A Cytosolic and nuclear fractions from HEYA8 and SKOV3. ip1 NM cells treated with or without leptomycin B (LMB) immunoblotted for p70 S6K ;
  • B Fluorescence imaging of HEYA8 and SKOV3. ip1 NM cells expressing GFP-p70 S6K after LMB treatment;
  • C Cytosolic and nuclear fractions from HEYA8 and SKOV3. ip1 HM cells treated with or without importazole immunoblotted for p70 S6K ;
  • D Fluorescence imaging of HEYA8 and SKOV3.
  • ip1 NM (lower panel) cells exogenously expressing WT p70 S6K or D 3 E-E 389 p70 S6K immunoblotted for total p70 S6K .
  • J Fluorescence imaging of HEYA8 NM cells expressing WT p70 S6K or D 3 E-E 389 after HGF treatment.
  • Fig. 3 Kinase activity is not required for p70 S6K nuclear localization.
  • A Total cell lysis of HEYA8 HM transfected with pEGFPC1, WT p70 S6K , or KD p70 S6K immunoblotted for indicated proteins;
  • B Fluorescence imaging of HEYA8 HM cells expressing GFP tagged WT p70 S6K or KD p70 S6K ;
  • C Cytosolic and nuclear fractions of HEYA8 HM cells expressing WT p70 S6K or KD p70 S6K immunoblotted for total p70 S6K ;
  • D Total lysis of HEYA8 NM cells and SKOV3.
  • E Fluorescence imaging of HEYA8 NM cells expressing GFP-p70 S6K after PF-4708671 treatment;
  • F Cytosolic and nuclear fractions or of HEYA8 NM cells and SKOV3.
  • FIG. 4 Nuclear p70 S6K induces EMT.
  • A Fluorescence images showing the subcellular localization of NLS-p70 S6K and NES-p70 S6K in HEYA8 NM cells.
  • B Scattering assay shows the phenotypes of empty vector pEGFPC1 and NLS-p70S6K overexpressing HEYA8 NM cell colonies. At least 50 colonies were counted in each experiment.
  • C Total cell lysis of HEYA8 NM transfected with pEGFPC1, NLS-p70 S6K , or NES-p70 S6K immunoblotted for indicated proteins.
  • hnRNPA2/B1 interacts with p70 S6K and mediates the nuclear p70 S6K induced EMT.
  • A Western blotting analysis the EMT markers expression and
  • B Scattering assays of HEYA8 HM cells expressing NS siRNA or hnRNPA2/B1 siRNA.
  • C Western blotting analysis of cytosolic and nuclear fractions of various human ovarian cancer cells using indicated antibodies.
  • D hnRNPA2/B1 IP or
  • E p70 S6K IP of HEYA8 HM cells, immunoblotted for p70 S6K and hnRNPA2/B1.
  • nonrelevant immunoglobulin G (IgG) IP was used as negative control; IPs [p70 S6K IP, nonrelevant immunoglobulin G (IgG) IP] of (F) HEYA8 NM and HM cells; (G) HEYA8 NM cells treated with or without HGF; (H) HEYA8 HM cells treated with or without rapamycin, immunoblotted for p70 S6K and hnRNPA2/B1. (I) Total cell lysis of HEYA8 NM transfected with the indicated siRNA or/and plasmid DNA immunoblotted for the indicated proteins.
  • FIG. 6 Nuclear p70 S6K regulates hnRNPA2/B1 alternative splicing p70 S6K pre-mRNA, enhancing the production of short isoforms h6a and h6c.
  • A Schematic of the different isoforms of p70 S6K derived from the splicing of alternative exons as indicated.
  • B RT-PCR analysis of the expression level of p70 S6K short isoforms h6a and h6c in HEYA8 and SKOV3. ip1 NM and HM cells.
  • C Western blot analysis of total cell lysis from HEYA8 NM cells exogenously expressing vector pEGFPC1 or h6a or h6c.
  • p70 S6K is a nucleocytoplasmic shuttling protein. We found phosphorylation but not activation of p70 S6K induces its trafficking into and accumulation in the nucleus. We further report its binding to hnRNPA2/B1 in the nucleus controls the generation of alternative spliced isoforms that are involved in epithelial-to-mesenchymal transition, a developmental mechanism that becomes hijacked by cancer cells for the migration, invasion and metastasis, in tumor progression.
  • hnRNPs are crucial in all aspects of RNA processes as it also impacts DNA methylation, histone acetylation, and microRNA processing, as the master regulators of gene expression, and that may provide a mechanistic rationale for the atypical high frequency of epigenetic modifications (more than half of all active alternative splicing events are changed in ovarian tumors) in ovarian cancer.
  • Nuclear localization of p70 S6K is positively correlated with high metastatic potential in ovarian cancer.
  • p70 S6K actively shuttles between cytoplasm and nucleus in a phosphorylation-dependent manner
  • p70 S6K continuously shuttles between cytoplasm and nucleus
  • p70 S6K localization was studied in cells treated with the CRM1-dependent nuclear export inhibitor, leptomycin B (LMB) .
  • LMB leptomycin B
  • Treatment with LMB for 3 hours resulted in significant nuclear translocation of p70 S6K in NM cells, which have low basal levels nuclear p70 S6K (Fig. 2A) .
  • Overexpression of GFP-tagged p70 S6K further corroborated the accumulation of p70 S6K in the nucleus after LMB treatment (Fig. 2B) .
  • Hepatocyte growth factor promotes ovarian cancer cell motility and invasion through activation of p70 S6K (Zhou HY and Wong AS, 2006) , we examined if HGF-induced phosphorylation could affect nuclear translocation of p70 S6K .
  • Phosphorylation of p70 S6K ( ⁇ 1.3 fold) could be detected within 15 min after HGF stimulation, and this increase closely correlated with p70 S6K translocation into the nucleus.
  • p70 S6K was maximally phosphorylated ( ⁇ 3 fold increase) by 60 min, at which p70 S6K nuclear accumulation was also maximized.
  • An increase of nuclear localization of ectopic GFP-p70 S6K was also observed upon HGF treatment (Fig. 2F) .
  • the mTOR inhibitor rapamycin, which could potently suppress p70 S6K phosphorylation.
  • rapamycin inhibited both p70 S6K phosphorylation and its nuclear localization in a dose-dependent manner, as observed by Western blotting of total cell lysates and subcellular fractions respectively (Fig. 2G) .
  • rapamycin abolished p70 S6K nuclear translocation in response to HGF (Fig. 2H) .
  • a kinase-dead (KD) mutant of p70 S6K Q100
  • KD kinase-dead
  • HEYA8 HM cells expressing KD p70 S6K displayed a reduction of S6 phosphorylation but not total S6 expression, compared with the cells expressing WT p70 S6K , confirming the lack of kinase activity of KD mutant (Fig. 3A) .
  • Fluorescence microscopy and subcellular fractionation studies of HM cells demonstrated that nuclear localization of KD p70 S6K was comparable to WT p70 S6K (Fig. 3B and C) . All these data indicate that the phosphorylation, but not activation, of p70 S6K is important for its nuclear accumulation.
  • p70 S6K is known to activate EMT, however, how its subcellular localization contribute to this function is still unknown. Therefore, we tagged p70 S6K with either a SV40 nuclear localization signal (NLS-p70 S6K ) or a protein kinase A inhibitor nuclear export signal (NES-p70 S6K ) at the N-terminal so that the exogenous p70 S6K could be confined to the nucleus and cytoplasm respectively. Nuclear localization of NLS-p70 S6K and cytosolic localization of NES-p70 S6K were confirmed by both microscopic observation (Fig. 4A) . We then performed scattering assay and examined the morphology of NLS-p70 S6K overexpressing cells.
  • NLS-p70 S6K SV40 nuclear localization signal
  • NES-p70 S6K protein kinase A inhibitor nuclear export signal
  • NM cells transfected with NLS-p70 S6K were more dispersed and exhibited more scattered colonies as compared to cells transfected with empty vector (Fig. 4C) .
  • HEYA8 NM cells lost expression of the epithelial marker E-cadherin.
  • the expression of mesenchymal markers, including N-cadherin and vimentin was strongly induced. Importantly, these alterations were to a slightly greater extent than NES-tagged p70 S6K , suggesting that nuclear import of p70 S6K is crucial for efficient EMT (Fig. 4D) .
  • Nuclear p70 S6K interacts with hnRNPA2/B1 to mediate EMT
  • hnRNPA2/B1 was co-immunoprecipitated by p70 S6K , but not by IgG control (Fig. 5F) .
  • p70 S6K could be co-immunoprecipitated with hnRNAPA2/B1 (Fig. 5G) .
  • Enhanced interaction between p70 S6K and hnRNPA2/B1 in HM than NM cells was also verified (Fig. 5H) .
  • HGF stimulation increased the p70 S6K -hnRNPA2/B1 association, which was suppressed in the presence of rapamycin (Fig. 5I and J) .
  • Table 1 The top three differentially precipitated proteins in HEYA8 HM and NM cells in a comparative and label-free quantitative LC-MS/MS.
  • HM cells transfected with hnRNPA2/B1 siRNA exhibited a suppressed mesenchymal phenotype, as evidenced by the reduction of N-cadherin, vimentin and the upregulation of E-cadherin as determined by Western blot (Fig. 5C) , and the more compact morphology of cell colonies (Fig. 5D) , suggestive of the involvement of hnRNPA2/B1 in EMT.
  • hnRNPA2/B1 siRNA was used in NLS-p70 S6K -overexpressing HEYA8 NM cells.
  • Nuclear p70 S6K /hnRNPA2/B1 upregulates EMT of ovarian cancer cells via modulating alternative splicing of RPS6KB1 pre-mRNA
  • HnRNPA2/B1 was shown to regulate alternative splicing by either direct binding to pre-mRNA or indirect regulation of spliceosome assembly and function.
  • RNA-IP RNA coimmunoprecipitation
  • hnRNPA2/B1 siRNA also significantly reduced the expression of h6a and h6c in HM cells.
  • nuclear translocation of p70 S6K promotes EMT provides new therapeutic insights for ovarian cancer anti-EMT therapies.
  • the level of nuclear p70 S6K was more pronounced for the highly metastatic experimental tumors and also in advanced stage clinical tumors, shows that nuclear p70 S6K is a prognostic indicator of ovarian cancer.
  • Our data showing that both cytoplasmic and nuclear p70 S6K promote EMT shows that p70 S6K regulates EMT via differential pathways in cytoplasm and nucleus.
  • the present disclosure shows the key roles of nuclear p70 S6K in ovarian tumor metastasis.
  • Our data demonstrate the modulation of p70 S6K regulates the metastatic potential of tumor cells. Since most cancer-related mortalities result from metastatic disease and no mutations that are selective for metastases have been identified. It is imperative to identify potential metastatic mediators for prognostic and therapeutic benefit.
  • the identification of nuclear p70 S6K , hnRNPA2/B1, and h6a and h6c provide additional targets that serve as predictive markers of metastasis, as well as effective targets for anti-metastatic therapies.
  • anti-cancer therapies it is important to selectively block nuclear p70 S6K activity while sparing its functions in other cellular compartments. Indeed, the selective inhibition of nuclear p70 S6K could avoid hyperglycemia: one of the most unwanted side effects derived from downregulation of cytoplasmic p70 S6K by pharmacological inhibitors.
  • ip1 were established as described before (To SKY et al., 2017) .
  • HEYA8, SKOV. ip1, SKOV3 and A2780 cells were routinely cultured in RPMI 1640 medium supplemented with 5%fetal bovine serum (Hyclone, Logan, UT) and 1%penicillin-streptomycin mixture (Invitrogen, Carlsbad, CA) in a humidified incubator with 5%CO 2 at 37°C.
  • CAOV3, OVCAR3, OVCAR429 were grown in a 1: 1 M199 and MCDB105 medium with the same additives, under the same conditions.
  • Mouse immunoglobulin G (catalogue number 1721011) was purchased from Bio-Rad Laboratories, rabbit IgG (catalogue number sc2027) was purchased from Santa Cruz.
  • Recombinant human hepatocyte growth factor (HGF) (catalogue number 294-HG-005) were purchased from R&D Systems.
  • Rapamycin (catalogue number 553210) and importazole (catalogue number SML0341) were purchased from Sigma-Aldrich.
  • Leptomycin B (LMB) (catalogue number L-6100) was purchased from LC Laboratories.
  • siRNA Small interfering RNA
  • siRNA targeting human hnRNPA2/B1 mRNA (target sequence, 5'-CGGUGGAAAUUUCGGACCA-3') (SEQ ID NO. 1) were synthesized by Dharmacon RNAi Technologies (Lafayette, CO) .
  • a NS siRNA was used as control (D-001210-01) .
  • Wildtype p70 S6K (WT p70 S6K ) , SV40 NLS-tagged p70 S6K (NLS-p70 S6K ) and PKI NES-tagged p70 S6K (NES-p70 S6K ) were subcloned in the pEGFPC1 expressing vector (Clontech, Palo Alto, CA) between the EcoR1 and the BamH1 sites.
  • the myc-tagged constitutively active p70 S6K (D 3 E-E 398 ) was a kind gift from Dr. George Thomas (Genome Research Institute, University of Cincinnati, Cincinnati, OH) (Jefferies et al., 1997) .
  • KD Kinase-dead
  • transient plasmid transfections were performed according to the standard Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA) protocol. All siRNAs were transfected into cells using siLentFect Lipid Reagent (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. Lipofectamine 2000 reagent was used when co-transfection of plasmid DNA and siRNA is necessary.
  • Cells were fractionated as described by Guillemin I et al. (2005) with little modification. Briefly, cells were harvested by scraping in CLB buffer (10mM HEPES, 10mM NaCl, 1mM KH 2 PO 4 , 5mM NaHCO 3 , 5 mM EDTA, 1 mM CaCl 2 , 0.5 mM MgCl 2 ) after being washed twice with phosphate-buffered saline pH 7.4 (PBS) . Cell suspension was homogenized with a glass homogenizer. The homogenate was centrifuged at 6,300 ⁇ g for 5 min at 4°C to sediment the crude nuclei.
  • CLB buffer 10mM HEPES, 10mM NaCl, 1mM KH 2 PO 4 , 5mM NaHCO 3 , 5 mM EDTA, 1 mM CaCl 2 , 0.5 mM MgCl 2
  • PBS phosphate-buffered saline pH 7.4
  • the supernatant was centrifuged at 17000 ⁇ g for 2 hours, and the resulting supernatant formed the cytoplasm fraction.
  • the crude nuclei were resuspended in TSE buffer (10 mM Tris, 300mM sucrose, 1 mM EDTA, 0.1%IGEPAL-CA 630 v/v, PH 7.5) and homogenized with a glass homogenizer. This suspension was centrifuged at 4,000 ⁇ g for 5 minutes. The resulting pellet was washed with TSE buffer twice and referred to as purified nuclei.
  • cytoplasmic and nuclear extracts or total lysis were separated using polyacrylamide gels and transferred to nitrocellulose membranes. After being blocked with 5%(weight/volume) non-fat dry milk (1 hour at room temperature) , the membranes were incubated with primary antibodies, washed, and further incubated with secondary antibodies conjugated with horseradish peroxidase. Followinged by another three 10-min washes in PBS + Tween 20 (PBST buffer) , protein bands were detected by chemiluminescence and their intensities were determined by Image J software.
  • PBST buffer PBS + Tween 20
  • RNA extraction and reverse transcription PCR (RT-PCR)
  • the primers used in this study are as follows: p70 S6K forward: 5-CTCTACCTCATCCTTGAGTATCTCAGTG-3’ (SEQ ID NO. 2) , reverse: 5-CATAGATTC ATACGCAGGTGC-3’ (SEQ ID NO. 3) ; E-cadherin forward: 5-GGGTGACTACAAAATCAATC-3’ (SEQ ID NO.
  • RNA-IP RNA-IP
  • the lysis buffer was prepared with DEPC-treated water and RNA inhibitor was added.
  • Primary antibody, or normal rabbit/mouse IgG (control) was added to the cleared lysates, and the mixture was incubated overnight at 4°C with agitation.
  • Protein A/G agarose beads were added into the mixture and binding was carried out for 4 h at 4°C. After being washed for four times with NP-40 buffer, for binding protein detection, the beads were eluted in 2 ⁇ sample laemmli buffer prior to Western blotting analysis; for binding RNA examination, the beads were suspended in Trizol reagent for RNA isolation and RT-PCR.
  • Cells were seeded on glass cover slips in 24 well plates 24 hours before transfection. 24 hours post-transfection, cells were washed three times with PBS and fixed with 4%paraformaldehyde for 20 minutes, permeabilized 0.1%Triton X-100 (Bio-Rad, Hercules, CA) , blocked with 5%bovine serum albumin for 30 minutes before being incubated with diluted primary antibody (1: 50 in PBS) . Cells were then washed and further incubated with Alexa Fluor 488 conjugated secondary antibodies (1: 200 in PBS) . Nuclei were stained with 4', 6-diamidino-2-phenylindole before mounting. Cells expressing GFP fusion proteins were stained with 4', 6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Cells were then observed and pictured by fluorescent microscopy (Nikon 80i Eclipse, Japan) .
  • Cells were seeded in 6-well tissue culture dishes at a density of 2 ⁇ 10 3 per well and allowed to form small colonies in 7 days. Cells were transfected with DNA plasmids or siRNA and grow for another 72 hours. Colonies were fixed with methanol, stained with crystal violet and observed under microscopy. The number of colonies maintained compact (50-90%of have cell-cell contact) was counted.
  • HEYA8 NM and HM cells were fractionated respectively for nucleus extraction. Each NM and HM nuclear sample was precipitated with p70 S6K antibody as described above. 1 ⁇ lithium dodecyl sulfate sample buffer (pH 8.5, 106 mm Tris-HCl, 141 mm Tris-Base, 2%LDS, 10%glycerol, 0.51 mm EDTA, 0.22 mm SERVA Blue G250, 0.175 mm Phenol Red for 4 ⁇ LDS) was used to eluted the precipitates. After centrifugation, reducing agent DTT was added (50 mM) into supernatant and incubated for 10 min at 70°C for proteins denature.
  • LC ⁇ MS/MS analyses were performed on a linear ion trap Oribtrap Velos mass spectrometer (LTQ Orbitrap Velos, Thermo-Scientific, Waltham, MA, USA) which is connected to a liquid chromatography (Finnigan, Thermo-Scientific) . Three micrograms of nuclear protein were loaded onto a column and rinsed with 2%buffer B at a flow rate of ⁇ 1000 nl/min for 8 min.
  • Tumor necrosis factor receptor-associated factor (TRAF) 4 is a new binding partner for the p70S6 serine/threonine kinase. Leuk Res. 27, 687-694 (2003) .
  • S6K ribosomal protein S6 kinase
  • Herpes simplex virus type 1 infection induces activation and recruitment of protein kinase C to the nuclear membrane and increased phosphorylation of lamin B. J Virol. 2006 Jan; 80 (1) : 494-504.

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Abstract

L'invention concerne une méthode de diagnostic ou de pronostic des métastases du cancer par la mesure de la p70S6K dans le noyau. L'invention concerne également une méthode de diagnostic des métastases du cancer par mesure de la liaison de la p70S6K à hnRNPA2/B1 dans le noyau. L'invention concerne une méthode de détection précoce de la dissémination péritonéale d'une tumeur ovarienne. L'invention concerne également une méthode de traitement du cancer.
PCT/CN2021/085641 2020-04-06 2021-04-06 Utilisation de la p70 s6 kinase nucléaire pour le diagnostic, le pronostic et le traitement du cancer WO2021204109A1 (fr)

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US9557338B2 (en) * 2012-10-18 2017-01-31 Signal Pharmaceuticals, Llc Inhibition of phosphorylation of PRAS40, GSK3-beta or P70S6K1 as a marker for tor kinase inhibitory activity
CN104388428A (zh) * 2014-10-22 2015-03-04 中国医科大学附属第一医院 一种干扰hnRNPA2/B1基因表达的双链siRNA及其应用

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