CN110064045B

CN110064045B - Application of micro-peptide CIP2A-BP in treating cancer

Info

Publication number: CN110064045B
Application number: CN201910403304.5A
Authority: CN
Inventors: 周翊峰; 邓杰琼; 郭宾宾; 张征; 吴思奇; 李华
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2019-05-15
Filing date: 2019-05-15
Publication date: 2022-12-23
Anticipated expiration: 2039-05-15
Also published as: CN110064045A

Abstract

The invention relates to application of a micro-peptide CIP2A-BP in treating cancer, and researches the influence of the micro-peptide on the metastasis and migration behavior of breast cancer cells from the perspective of the micro-peptide coded by lncRNA at the translation level. And through the treatment of targeting micro peptide CIP2A-BP, the micro peptide CIP2A-BP and B56 gamma subunit of PP2A are competitively combined with cell oncogene CIP2A, so that PP2A activity is released, and the activation of PI3K/AKT/NF kappa B pathway is inhibited, resulting in the reduction of the expression level of matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and EMT induced transcription factor Snail. In vitro and in vivo experiments prove that the micro-peptide CIP2A-BP can obviously inhibit the metastasis and invasion of breast cancer cells and has clinical application value on breast cancer.

Description

Application of micro-peptide CIP2A-BP in treating cancer

Technical Field

The invention relates to application of a micro-peptide CIP2A-BP in treating cancer, belonging to the field of biological medicine.

Background

Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer, accounting for 15% of all breast cancers. TNBC is negative for Estrogen Receptor (ER) and Progesterone Receptor (PR) expression, lacking HER2 amplification or overexpression. It is often associated with more aggressive tumor behavior, including a smaller age of onset, shorter time to recurrence, higher risk of local and distant metastasis, and thus lower overall survival compared to other breast cancer subtypes. Currently, there is no effective targeted treatment for TNBC.

The TGF- β signaling pathway plays an important role in the development and growth of normal cells. Alterations in the TGF-. Beta.signaling pathway have been reported in tumorigenesis, tumor invasion and metastasis. In breast cancer, TGF- β treatment can induce Epithelial Mesenchymal Transition (EMT), inducing cancer stem cells, while TGF- β inhibition can enhance chemotherapy of TNBC.

Long non-coding RNA (lncRNA) is an RNA longer than 200nt, does not normally encode a protein, and is involved in normal cellular functions and disease progression by regulating transcription or translation. Recent studies have shown that some of these lncrnas encode bioactive micro-peptides that are involved in a variety of cellular activities. Previous studies have shown that TGF- β treatment can directly regulate lncrnas at the transcriptional level, however, few know whether TGF- β treatment can directly affect lncrnas at the translational level.

Reports indicate that many lncrnas regulate biological pathways and processes by encoding and translating micro-peptides. For example, LINC00961 encodes a SPAR peptide that down-regulates mTORC1 activation and muscle regeneration; lncRNA HOXB-AS3 encodes a 53-aa peptide that inhibits the growth of colon cancer; lncRNA-Six1 encodes a 7.26kDa mini-peptide that activates the Six1 gene to promote cell proliferation and muscle growth; the muscle regulatory protein is a skeletal muscle physiological regulator encoded by skeletal muscle specific LncRNA.

Disclosure of Invention

The invention aims to provide LINC00665 coded functional micro peptide CIP2A-BP which serves as a tumor suppressor gene in TNBC and can effectively suppress breast cancer cells.

In order to achieve the purpose, the invention provides the following technical scheme: the application of the micro-peptide CIP2A-BP or pharmaceutically acceptable salt thereof in preparing a medicament for treating cancer, wherein the cancer is breast cancer, and the amino acid sequence of the micro-peptide CIP2A-BP is shown as SEQ ID NO. 1.

The invention also provides application of a pharmaceutical composition containing the micro-peptide CIP2A-BP or pharmaceutically acceptable salt thereof in preparing a medicament for treating cancer, wherein the cancer is breast cancer, and the amino acid sequence of the micro-peptide CIP2A-BP is shown as SEQ ID NO. 1.

Further, the breast cancer is triple negative breast cancer.

Further, the mini-peptide CIP2A-BP is encoded by LINC00665 gene.

Further, the sequence of the LINC00665 gene is shown in SEQ ID NO. 2.

Further, the medicament has at least one of the following functions:

1) Reducing the incidence of chemical-induced breast cancer;

2) Slowing or stopping the growth of established breast cancer foci;

3) Slowing or stopping metastasis of established breast cancer foci;

4) Inducing CTL cell with breast cancer specificity and killing to breast cancer cell.

Further, the mini-peptide CIP2A-BP or a pharmaceutically acceptable salt thereof is used alone as an active ingredient, or the mini-peptide CIP2A-BP or a pharmaceutically acceptable salt thereof is used in combination with an additional pharmaceutically active compound.

Furthermore, the pharmaceutical composition also contains pharmaceutically acceptable auxiliary materials.

The invention has the beneficial effects that: according to the invention, from the perspective of translating the horizontal lncRNA-encoded micro-peptide, the influence of the micro-peptide on the malignant biological behaviors of breast cancer cell metastasis and migration is researched, and the important function of the micro-peptide in breast cancer is proved. In view of the inhibition of the micro-peptide CIP2A-BP on the metastasis and invasion of breast cancer cells, the means for inhibiting the tumor development is formed by changing the expression quantity of the micro-peptide and further changing the activity of PP2A to influence the PI3K/AKT/NF kappa B pathway. And, the inventor leads the micro peptide CIP2A-BP to competitively bind with the cell oncogene CIP2A with the B56 gamma subunit of PP2A through the treatment of the targeting micro peptide CIP2A-BP, thereby releasing the activity of PP2A, inhibiting the activation of PI3K/AKT/NF kappa B pathway, leading to the reduction of the expression level of matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and EMT induced transcription factor Snail. In vitro and in vivo experiments prove that the micro-peptide CIP2A-BP can obviously inhibit the metastasis and invasion of breast cancer cells and has clinical application value on breast cancer.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

Fig. 1A to fig. 1H are experimental data and schematic diagrams of the effect of the micro-peptide CIP2A-BP on the migration and invasion of triple negative breast cancer cells in vitro and in vivo according to the first embodiment of the present invention, specifically:

fig. 1A and 1B: migration and invasion TNBC cells were transfected with LINC00665 ORF and control lentivirus for the indicated times, right panel is the quantification of TNBC cells, which migrated through the membrane without Matrigel (top) and invaded through Matrigel-coated membrane (bottom);

fig. 1C and 1D: migration and invasion of wild-type and CIP2A-BP knock-out TNBC cells and CIP2A-BP re-express Hs578TKO and MDA-MB-231KO cells, right panel is a quantification of cells that migrated through the membrane without Matrigel (top) and invaded through Matrigel-coated membrane (bottom);

FIG. 1E: wound healing assay of Hs578T and MDA-MB-231 cells transfected with the LINC00665 ORF for expression and control of lentivirus and photographing the wound margins at the indicated time points after trauma, right panel is quantification of relative wound healing area;

FIG. 1F: wild-type and CIP2A-BP knock-out TNBC cells and re-expressed CIP2A-BP Hs578TKO and MDA-MB-231KO were seeded on cell culture inserts for wound healing assays and wound margins were photographed at the indicated time points after wounding, right panel is quantification of relative wound healing area;

fig. 1G and fig. 1H: upper part: by tail vein injection, indicated TNBC cells (1.0X 10) ⁵ -10 ⁶ Cell/mouse) injected nude mice: lung metastases were observed 8 weeks after injection (n = 5); and (3) the method is low: histological analysis of lung metastasis in a mouse model was shown by Hematoxylin and Eosin (HE) staining.

Fig. 2A to 2F are experimental data graphs and schematic diagrams illustrating the effect of the micro-peptide CIP2A-BP on the metastasis and invasion of breast cancer cells in the MMTV-PyMT mouse model in example two of the present invention, specifically:

FIG. 2A: upper part: lung metastatic nodules were observed from 140 day old MMTV-PyMT mice (n = 5); and (3) the method is low: histological analysis of lung metastasis in mouse models by Hematoxylin and Eosin (HE) staining;

FIG. 2B: from MMTV-PyMT; CIP2A-BP +/+ or MMTV-PyMT; immunohistochemical (IHC) staining of p-AKT of mammary tumors of CIP 2A-BP-/-mice, lower panel shows higher magnification image of inset in upper panel.

FIG. 2C: upper part: MMTV-PyMT mice were injected with CIP2A-BP or svCIP2A-BP via mammary gland fat pads; lung metastatic nodules were observed in 140-day-old MMTV-PyMT mice (n = 5); and (3) the method is low: histological analysis of lung metastases in mouse models by Hematoxylin and Eosin (HE) staining;

FIG. 2D: representative IHC images of p-AKT from breast tumors of MMTV-PyMT mice injected with CIP2A-BP or svCIP2A-BP by breast fat pad; the lower panel displays a higher magnification image of the inset in the upper panel;

FIG. 2E: kaplan-Meier survival curves of nude mice injected with MDA-MB-231 cell CIP2A-BP or svCIP2A-BP, the horizontal line indicates the time after the mice were injected with cells via tail vein, and the number of test mice per group was 10;

FIG. 2F: upper part: nude mice injected with MDA-MB-231 cells and CIP2A-BP or svCIP2A-BP via tail vein; metastatic nodules of the lung were observed 8 weeks after injection (n = 5); and (3) the method is low: histological analysis of lung metastases in mouse models was shown by Hematoxylin and Eosin (HE) staining.

FIGS. 3A and 3B are Kaplan-Meier overall survival curves for TNBC patients with the mini-peptide CIP2A-BP of example III of the present invention.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Aiming at the condition that the treatment means for breast cancer is limited at present, the applicant researches and explores the treatment value of the micro-peptide CIP2A-BP for the breast cancer from the perspective of translating the micro-peptide coded by lncRNA at the horizontal level. The invention firstly identifies the long non-coding RNA LINC00665 of the coding micro-peptide CIP2A-BP, and the coding frame is ORF1. It was next demonstrated that TGF- β activated Smad signaling pathway induces the expression of translational suppressor 4E-BP1 in triple negative breast cancer cell lines, which inhibits the eukaryotic translation initiation factor elF4E, resulting in reduced translation of CIP2A-BP from LINC 00665. Thus, the mini-peptides were down-regulated by TGF- β treatment in breast cancer cell lines. In addition, in vitro knock-out and overexpression studies demonstrated that CIP2A-BP, but not LINC00665 transcript, acts as a tumor suppressor in breast cancer development and progression. Then, CIP2A-BP directly combines tumor oncogene CIP2A to replace B56 gamma subunit of PP2A, thereby releasing PP2A activity for inhibiting PI3K/AKT/NF kappa B pathway activation, and further influencing the expression of downstream targets, including MMP2, MMP9 and Snail. In conclusion, experiments found that TGF- β down regulates CIP2A-BP translation via CIP2A/PP2A and PI 3K/AKT/nfkb signaling pathways to induce tumor invasion and metastasis in triple negative breast cancers.

In some embodiments, a pharmaceutical composition containing the mini-peptide CIP2A-BP or a pharmaceutically acceptable salt thereof may be employed, which may optionally further comprise one or more additional pharmaceutically active compounds.

According to the invention, the pharmaceutical composition comprises the micro-peptide CIP2A-BP of the invention and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition can be administered, for example, orally, by injection, etc. The pharmaceutical composition of the present invention can be prepared into various dosage forms including, but not limited to, tablets, capsules, solutions, suspensions, granules, injections, etc. according to conventional methods in the art.

EXAMPLE A Micropeptide CIP2A-BP inhibits the migration and invasion of triple negative breast cancer cells

Transwel cell migration and invasion assay: the capacity of cell migration and Invasion was evaluated using a Corning Transwell insertion Chamber (Corning) with an 8mm size well and a BioCoat matrix Invasion Chamber (BD Biosciences), respectively. About 1.0X 10 in 200. Mu.l serum-free DMEM medium ⁴ (migration assay) or 2.0X 10 ⁵ (invasion assay) transfected cells were seeded in the upper well; the chamber was then incubated with medium plus 20% fetal bovine serum at 37 ℃ for 48 hours to allow the cells to migrate to the lower well. Cells that have migrated or invaded the membrane are fixed in methanol, stained with crystal violet, imaged and counted.

Wound healing experiments: cells were seeded on ibidi cell culture inserts in 35-mm culture dishes and 5% CO at 37% ₂ Culturing in medium. After 24 hours, the culture was removedInsert and add media. Wound healing within the gap was observed at different time points and wound closure was assessed in five random fields using an inverted microscope. Gaps were analyzed using Image J software and each experiment was repeated three times.

Transwell and wound healing assays using TNBC cells with CIP2A-BP overexpression or knock-out found that the mini-peptide CIP2A-BP plays a role in the progression of TNBC. Referring to fig. 1A-1F, it was experimentally found that overexpression of the His-tagged LINC00665 ORF in TNBC cell line Hs578T and MDA-MB-231 reduced migration and invasion in the Transwell assay (fig. 1A and 1B), and migration in the wound healing scratch assay (fig. 1E and 1F). In contrast, CIP2A-BP knockdown in Hs578T and MDA-MB-231 cell lines increased migration and invasion in the Transwell assay (fig. 1C and 1D), and migration in the wound healing scratch assay (fig. 1E and 1F). This increase in migration and invasion can be reversed by over-expressing the LINC00665 ORF in the Hs578TKO and MDA-MB-231KO cell lines (FIGS. 1C-1F). These results indicate that CIP2A-BP plays an important role in TNBC migration and invasion.

Referring to FIGS. 1G and 1H, the experiment was followed to determine the effect of LINC00665 ORF overexpression and CIP2A-BP knockout on tumor metastasis using a mouse TNBC lung metastasis xenograft model. By tail vein injection, indicated TNBC cells (1.0X 10) ⁵ -10 ⁶ ) Injecting a nude mouse; lung metastases were observed 8 weeks after injection. It was found that LINC00665 ORF overexpressing TNBC cell line produced a lower number of lung metastatic nodules than wild type TNBC cell line, while CIP2A-BP knock-out TNBC cell line produced a higher number of lung metastatic nodules. The experimental result shows that the micro-peptide CIP2A-BP encoded by LINC00665 plays a role of an inhibitor for cell migration and invasion in TNBC.

Example two-Micropeptide CIP2A-BP inhibits Breast cancer metastasis and invasion in MMTV-PyMT mouse model

Referring to FIGS. 2A-2F, CIP2A-BP was first transferred to C57BL/6 mice and then mated with MMTV-PyMT mice to produce MMTV-PyMTCIP2A-BP +/+ mice. MMTV-PyMTCIP2A-BP +/+ mice were found to have less lung metastases compared to MMTV-PyMT mice. And the primary tumor compared with MMTV-PyMT is found through experimentsMMTV-PyMTCIP2A-BP +/+ mice had lower p-AKT expression. First, CIP2A-BP was transferred into C57BL/6 mice using MM, and then mated with MMTV-PyMT mice to produce MMTV-PyMT; CIP2A-BP +/+ mice. It was found that compared to MMTV-PyMT mice, MMTV-PyMT; CIP2A-BP +/+ mice had less lung metastasis (FIG. 2A). Furthermore, the primary tumor compared with MMTV-PyMT, MMTV-PyMT; CIP2A-BP +/+ mice had lower p-AKT expression (FIG. 2B). The effect of exogenous CIP2A-BP treatment on lung metastasis was further investigated using the MMTV-PyMT mouse model. CIP2A-BP (15 mg/kg) was injected into mammary fat pads of 8-week-old MMTV-PyMT mice. On day 7 post-injection, 5 mice per group were re-injected once weekly with CIP2A-BP (15 mg/kg) for an additional 5 weeks. Metastases in the lungs were directly observed in randomly selected fields 12 weeks after injection. As a result, breast fat pad injection of CIP2A-BP significantly reduced the number of lung metastatic nodules (FIG. 2C). At the same time, it was found that exogenous CIP2A-BP treatment significantly reduced the p-AKT level of the primary tumor. In summary, CIP2A-BP was found experimentally to inhibit AKT phosphorylation in primary tumors and lung metastases in the MMTV-PyMT mouse model (fig. 2D). In addition, to determine the antitumor activity of the mini-peptides in vivo, MDA-MB-231 cells (1.0X 10) were tested ⁵ -10 ⁶ ) And CIP2A-BP (300. Mu.g/ml) was injected into the tail vein of the mice. On day 7 post-injection, CIP2A-BP (15 mg/kg) was re-injected into the tail vein of the mice. Results mice with CIP2A-BP injection had significantly improved survival compared to control mice (FIG. 2E). At the same time, tissue HE staining indicated that CIP2A-BP also significantly reduced the number of lung metastases (FIG. 2F). Therefore, in vivo experiments prove that the micro-peptide CIP2A-BP can obviously inhibit the metastasis and invasion of breast cancer cells and improve the survival rate.

Example Low expression of the Trinanopeptide CIP2A-BP is associated with poor survival in patients with triple negative breast cancer

Experiments confirmed that levels of the mini-peptide CIP2A-BP correlate with overall survival in TNBC patients. Using median expression levels of TNBC patients, CIP2A-BP, experiments divided TNBC patients into two different groups: high CIP2A-BP patients (relative expression level > median expression level) and low CIP2A-BP patients (relative expression level ≦ median expression) levels), first cohort (suzhou cohort) and second cohort (guangzhou cohort). The log rank test and Kaplan-Meier survival curves were used, see FIG. 3A and FIG. 3B. The experimental results found that in the first cohort, patients with low expression of CIP2A-BP had a significantly lower OS than patients with high expression of CIP2A-BP [ Median Survival Time (MST): 29vs 43 months, log rank P =0.02, risk ratio (HR) =2.64] and second cohort (MST: 26vs 42 months, log rank P =0.01, HR = 2.76).

In summary, the following steps: according to the invention, from the perspective of translating the horizontal lncRNA-encoded micro-peptide, the influence of the micro-peptide on the malignant biological behaviors of breast cancer cell metastasis and migration is researched, and the important function of the micro-peptide in breast cancer is proved. In view of the inhibition of the micro-peptide CIP2A-BP on the metastasis and invasion of breast cancer cells, the means for inhibiting the tumor development is formed by changing the expression quantity of the micro-peptide and further changing the activity of PP2A to influence the PI3K/AKT/NF kappa B pathway. And, the inventor leads the micro peptide CIP2A-BP to competitively bind with the cell oncogene CIP2A with the B56 gamma subunit of PP2A through the treatment of the targeting micro peptide CIP2A-BP, thereby releasing the activity of PP2A, inhibiting the activation of PI3K/AKT/NF kappa B pathway, leading to the reduction of the expression level of matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and EMT induced transcription factor Snail. In vitro and in vivo experiments prove that the micro-peptide CIP2A-BP can obviously inhibit the metastasis and invasion of breast cancer cells and has clinical application value on breast cancer.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Suzhou university

<120> use of the micropeptide CIP2A-BP for the treatment of cancer

<130> 0

<160> 2

<210> 1

<211> 52

<212> PRT

<213> Micropeptide (CIP 2A-BP)

<400> 1

Met Gly Arg Trp Arg Val Ser Ser Val Glu Ser Trp Pro Phe Ala

5 10 15

Ser Thr Gly Gly Lys Leu Ala Ile Ala Thr Ala Gly Thr Gly Thr

20 25 30

Gln Ala Ala Pro Arg Pro Phe Leu Ile Arg Pro Pro Asp Ser Trp

35 40 45

Ala Leu Ala Leu Ala Leu Met

50

<210> 2

<211> 159

<212> RNA

<213> Gene (LINC 00665)

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augggacgcu ggagggucag cagcguggag uccuggccuu uugcguccac gggugggaaa 60

uuggccauug ccacggcggg aacugggacu caggcugccc cccggccguu ucucauccgu 120

ccaccggacu cgugggcgcu cgcacuggcg cugauguag 159

Claims

1. The application of the micro-peptide CIP2A-BP in preparing the medicines for treating the cancer is characterized in that the cancer is triple negative breast cancer, and the amino acid sequence of the micro-peptide CIP2A-BP is shown as SEQ ID NO. 1.

2. Application of a pharmaceutical composition containing a micro-peptide CIP2A-BP in preparing a medicament for treating cancer, wherein the cancer is triple negative breast cancer, and the amino acid sequence of the micro-peptide CIP2A-BP is shown as SEQ ID NO. 1.

3. The use according to claim 1 or 2, wherein the mini-peptide CIP2A-BP is encoded by the LINC00665 gene of the sequence shown in SEQ ID No. 2.

4. The use according to claim 1 or 2, wherein the medicament has the following functions:

slowing the metastasis of established breast cancer foci.

5. The use according to claim 1, wherein the mini-peptide CIP2A-BP is used as the sole active ingredient or the mini-peptide CIP2A-BP is used in combination with an additional pharmaceutically active compound.

6. The use of claim 2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.