Mechanism And Role Of PTPRD In The Biological Behavior And Stemness Of Breast Cancer

Breast cancer is the most common malignancy worldwide. The death incidence of breast cancer has declined over time, due to the improvements in early diagnosis, advanced surgical techniques and combined therapy(surgery, chemotherapy and radiotherapy and molecular target treatment). Metastasis, the spread of cancer from the primary tumor to remote sites, is the major cause of fatality, resulting in about 90% of the deaths.The process of metastasis encompasses release of cells from the primary tumor site, invade into and transport of the cells in the blood vessel or laymphatic system, attachment to the remote site, and invasion and colonization of secondary site. In the process of tumor metastasis, tumor cells tend to undergo epithelial-mesenchymal transition(EMT). Therefore, it is very importance to understand the mechanisms that facilitate the invasive transition in breast cancer and the complex processes that underlie changes in cell migration and cell invasion. In recent years, studies suggest that cancer stem cells may play an important role in tumor metastasis.Cancer stem cells are a small fraction of tumor cells with stem cell properties, which can not only undergo self-renew but also generate different progeny. Accumulating evidence indicates that CSCs play a key role in not only the original tumorigenicity but also in their ability for local invasion and migration. CSCs has been isolated in many cancers such as lung cancer, colorectal cancer, glioblastoma, prostate cancer, lung cancer and other tumors. Breast cancer stem cells(BCSCs) is one of the earliest CSCs that been isolated from the solid tumor. CD44 and CD24 are the most commonly used BCSCs marker molecules. As few as 100 cells beating this phenotype were able to produce tumors in immune deficient NOD/SCID mice whereas over 100-fold greater cells that did not bear this phenotype was non-tumorigenic. More recently, it has been shown that both normal and malignant mammary stem/progenitor cells express high level of enzyme aldehyde dehydrogenase(ALDH), which can be assessed by the Aldefluor assay(Stem Cell Technologies). ALDH1+ cells have a strong ability of self-renewal and tumor formation planting. Overlapping with some features of normal stem cells, CSCs are shown to be resistant to proapoptotic factors, rendering them a formidable adversary to current anti-cancer modalities.Receptor-type tyrosine-protein phosphatase δ(PTPRD) is a receptor type tyrosine-protein phosphatase(PTPR). There are 21 members in the family of PTPRs, according to the extracellular domain will be divided into eight subfamilies. PTPRD belong LAR like PTPR family, which including PTPRD, PTPRS and LAR. PTPRD is composed of a cell adhesion molecule-like extracellular domain and cytoplasmic protein tyrosine phosphatase(PTP) domains. Full length of PTPRD extracellular region containing three immunoglobulin-like domains and eight FN-III-like domain, is connected to intracellular region through the transmembrane region peptide, intracellular region has two tyrosine phosphatase D1 and D2. These domains are all highly conserved. The phosphatase domain D1 proximal to the membrane, possesses >99% of the total catalytic activity, whereas D2, which is proximal to the C-terminal area, only binds several docking proteins.PTPRD is most commonly expressed in the central nervous system, where it is concentrated in growth cones of elongating processes. PTPRD is a homophilic cell adhesion molecule. These homophilic interactions serve to promote neuronal adhesion and neurite outgrowth. PTPRD has been suggested to be a tumour suppressor due to its inactivation in a number of human cancers, including head and neck, melanoma, lung cancer and neuroblastoma. Multiple mutations have been identified in tumours that may compromise not only activity, but also the function of the extracellular segment. Chromosome 9p, which harbours PTPRD gene, is also a frequent target of microdeletion in primary tumours and is subject to chromosome shedding in 6% of tumours studied.The phosphorylation state of tyrosine residues on proteins is fundamentally important for the control of different functions of the cell and is the result of the balance between protein tyrosine kinase(PTK) and protein tyrosine phosphatase(PTP) activities. Several studies has reported that PTPRD can dephosphorylate P-STAT3 and inhibit its activity. STAT3 is an oncogene, as a signal transducer and activator of transcription factor, can promote tumorigenesis, facilitate metastaisis, promote proliferation and inhibit apoptosis.In the present study, first, we studied the role of PTPRD in the migration and invasion of breast cancer cell line. Second, we studied the role of PTPRD in the stemness of BCSCs. Third, we analyzed the relationship of PTPRD with the IL-6-STAT3 signal pathway. Then we investigated the expression of PTPRD and P-STAT3 in the human breast cancer tissues. The role of PTPRD in breast cancer metastasis and stemnss were fully examined in present study.Part 1 PTPRD si RNA impact on breast cancer cell migration and invasionObjective: Investigate effect of PTPRD downregulation on breast cancer cell migration and invasion, analyze the relationship of PTPRD with the protein related to epithelial mesenchymal transformation.Methods: PTPRD was knockdown by two distinct small interference RNAs(si RNAs) in MDA-MB-231 breast cancer cell line. The migration and invasion ability were detected by scratch test and transwell chamber model. The expression of EMT-associated protein E-cadherin and Vimentin was detected by Western Blot(WB). The apoptosis rate induced by paclitaxel was investigated by flow cytometry. Cell proliferation level was detected by CCK-8, Cell cycle was detected by flow cytometry.Results: The cell transfected by PTPRD si RNA Scratch test and Transwell chamber model were found transfected cell migration and invasion PTPRD si RNA significantly higher than NC group unrelated sequence. After the cells were transfected PTPRD si RNA, which E-cadherin protein expression was significantly down-regulated, and Vimentin was significantly down-regulated. Under chemotherapy drugs paclitaxel, PTPRD si RNA group was significantly lower than the level of apoptosis NC group. PTPRD si RNA transfected cells did not differ in the level of the cell cycle and cell proliferation and NC.Conclusion: Downregulation of PTPRD can promote breast cancer cell lines MDA-MB-231 migration and invasion, and promote EMT. PTPRD downregulation can inhibit apoptosis, but has no significant effect on the proliferation and cell cycle.Part II PTPRD expression in BCSCs and it’s role in the stemness of BCSCsObjective: Study the PTPRD and related molecules STAT3, P-STAT3, EMT related molecule E-cadherin and Vimentin expression in breast cancer stem cells. Study PTPRD downward effect on breast cancer stem cell characteristics. Methods: Immunomagnetic beads were used to isolate the CD44+/CD24- breast cancer stem cells from serum-free suspension cultured breast cancer cells. The quantitative Real time PCR was used to detect the expression of PTPRD and STAT3. The protein expression of PTPRD, P-STAT3, STAT3, ALDH1, OCT-4, Vimentin and E-cadherin level were detected by WB. In order to detect the effect of downregulation of PTPRD on the stemness of MDA-MB-231, the PTPRD was knockdown by two distinct small interference RNAs(si RNAs), the holocolony forming ability was detected by Colony formation assay, the proportion of CD44+/CD24- breast cancer stem cells was detected by flow cytometry, mammosphere formation assay was used to detect the self-renewal ability of breast cancer stem cell. The stem cell marker ALDH1 and OCT-4 were detected by WB. PTPRD sh RNA infected cell lines were inoculated into nude mice, and monitored the growth of xenografts. Immunohistochemical staining and WB analysis were used to detect the expression of related proteins.Results: Downregulation of PTPRD premoted the proliferation and holoclone ability. The proportions of breast cancer stem cells were increased in the cell transfected by PTPRD si RNA comparing NC. The numbers of mammosphere increased in PTPRD si RNA transfected cells. The protein expression level of PTPRD was significantly downregulated in stem cells. The expression of Vimentin increased and E-cadherin decreased in cancer stem cells. The stem cell marker ALDH1, OCT-4 significantly increased in the cancer stem cells. While there was no difference in the expression of P-STAT3 in stem cells and nonstem cells. The tumor volume of xenografts in PTPRD sh RNA cells were big, grow rapidly. The expression level of PTPRD decreased and P-STAT3 increased in the xenografts forming by the cell transfected by PTPRD sh RNA transfected cells.Conclusion: ALDH1 and OCT-4 expression in CD44+/CD24- cell population significantly increased, indicating that we have successfully isolated the stem cells. The cancer stem cell has the mesenchymal cell characteristics. Downregulaion of PTPRD can promote the stemness, self-renewal ability and tumorgenesity of breast cancer stem cells. Breast cancer stem cell has the mesenchymal phenotype.Part III Mechanism of PTPRD in breast cancerObjective: Study the role of IL-6 signal transduction pathway in breast cancer cell migration and invasion. Investigate the effect of PTPRD downregulation on the activation of STAT3. Detect the expression of PTPRD during the activation of STAT3 by IL6.Methods: STAT3 and P-STAT3 expression levels were detected by WB. Proteins including PTPRD, STAT3, P-STAT3, Vimentin, E-cadherin, ALDH1 and OCT-4 were detected by WB in the MDA-MB-231 cells collected in 1h, 3h, 6h, 12 h, 24 h, 48 h after IL-6 action. Wound healing assay was used to detect the role of IL-6 on cell migration, CCK8 were used to analyze the proliferation ability. Results: IL-6 promote the migration and invasion of breast cancer cells. WB showed that P-STAT3 expression increased in 1 hour and peak in about 3 hour. PTPRD protein level decreased immediately after IL-6 exposure along with the activation of STAT3(Fig 1). The cells exposed to IL-6 have higher level expression of stem cell markers ALDH1 and OCT-4. Treatment of MDA-MB-231 cells with IL-6 resulted in EMT, as evidenced by induction of the mesenchymal markers Vimentin and repression of the epithelial marker E-cadherin.Conclusion: IL-6 induces EMT, invasiveness, and migration properties of breast cancer calls. Downregulation of PTPRD induced the activation of STAT3. Treatment of DLD-1 cells with IL-6 resulted in activation of P-STAT3 accompanied by the increased expression of PTPRD.Part IV PTPRD, P-STAT3 expression in breast cancers and their relationship with clinicopathological parameters and prognosisObjective: The current study aimed to investigate PTPRD and P-STAT3 expression in breast cancer, there relationship with the clinicopa parameters and their prognostic significance in breast cancers.Methods: Immunohistochemistry were used to examine PTPRD and P-STAT3 expression in breast cancer and relatively normal breast tissues. Association between PTPRD and P-STAT3 expression and the clinical pathological features was analyzed. Kaplan-Meier analysis was performed to analyze the correlation between the PTPRD and P-STAT3 alterattion and prognosis.Results:In 76 cases of breast cancer, 48 cases showed PTPRD weak expression and 38 cases showed overexpression. Twenty two cases showed P-STAT3 weak expression and 54 cases showed overexpression. Reduced PTPRD expression was significantly associated with tumor size and ER expression. Increased P-STAT3 expression was significantly associated with tumor grade. Kaplan-Meier survival analysis. revealed that low expression of P-STAT3 significantly correlated with poor survival of breast cancer patients(P<0.05).Conclusion: These results indicated that PTPRD is a candidate tumour suppressor in breast cancer. Activation of STAT3 promotes breast cancer tumorgenesis and metastasis. P-STAT3 may be a prognosis predict factor.