Loss of PTPN12 Stimulates Progression of ErbB2-Dependent Breast Cancer by Enhancing Cell Survival,Migration, and Epithelial-to-Mesenchymal Transition

PTPN12 is a cytoplasmic protein tyrosine phosphatase (PTP) reported to be a tumor suppressor in breast cancer, through its capacity to dephosphorylate oncogenic receptor protein tyrosine kinases (PTKs), such as ErbB2. However, the precise molecular and cellular impact of PTPN12 deficiency in breast cancer progression remains to be fully clarified. Here, we addressed this issue by examining the effect of PTPN12 deficiency on breast cancer progression in vivo, in a mouse model of ErbB2-dependent breast cancer using a conditional PTPN12-deficient mouse. Our studies showed that lack of PTPN12 in breast epithelial cells accelerated breast cancer development and lung metastases in vivo. PTPN12-deficient breast cancer cells displayed enhanced tyrosine phosphorylation of the adaptor Cas, the adaptor paxillin, and the kinase Pyk2. They exhibited no detectable increase in ErbB2 tyrosine phosphorylation. PTPN12-deficient cells were more resistant to anoikis and had augmented migratory and invasive properties. Enhanced migration was corrected by inhibiting Pyk2. PTPN12-deficient breast cancer cells also acquired partial features of epithelial-to-mesenchymal transition (EMT), a feature of more aggressive forms of breast cancer. Hence, loss of PTPN12 promoted tumor progression in a mouse model of breast cancer, supporting the notion that PTPN12 is a tumor suppressor in human breast cancer. This function was related to the ability of PTPN12 to suppress cell survival, migration, invasiveness, and EMT and to inhibit tyrosine phosphorylation of Cas, Pyk2, and paxillin. These findings enhance our understanding of the role and mechanism of action of PTPN12 in the control of breast cancer progression.     

Mitotic kinases as drivers of the epithelial-to-mesenchymal transition and as therapeutic targets against breast cancers

Biological therapies against breast cancer patients with tumors positive for the estrogen and progesterone hormone receptors and Her2 amplification have greatly improved their survival. However, to date, there are no effective biological therapies against breast cancers that lack these three receptors or triple-negative breast cancers (TNBC). TNBC correlates with poor survival, in part because they relapse following chemo- and radio-therapies. TNBC is intrinsically aggressive since they have high mitotic indexes and tend to metastasize to the central nervous system. TNBCs are more likely to display centrosome amplification, an abnormal phenotype that results in defective mitotic spindles and abnormal cytokinesis, which culminate in aneuploidy and chromosome instability (known causes of tumor initiation and chemo-resistance). Besides their known role in cell cycle control, mitotic kinases have been also studied in different types of cancer including breast, especially in the context of epithelial-to-mesenchymal transition (EMT). EMT is a cellular process characterized by the loss of cell polarity, reorganization of the cytoskeleton, and signaling reprogramming (upregulation of mesenchymal genes and downregulation of epithelial genes). Previously, we and others have shown the effects of mitotic kinases like Nek2 and Mps1 (TTK) on EMT. In this review, we focus on Aurora A, Aurora B, Bub1, and highly expressed in cancer (Hec1) as novel targets for therapeutic interventions in breast cancer and their effects on EMT. We highlight the established relationships and interactions of these and other mitotic kinases, clinical trial studies involving mitotic kinases, and the importance that represents to develop drugs against these proteins as potential targets in the primary care therapy for TNBC.     

ErbB2, EphrinB1, Src kinase and PTPN13 signaling complex regulates MAP kinase signaling in human cancers

In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression.     

The lysosomal TRPML1 channel regulates triple negative breast cancer development by promoting mTORC1 and purinergic signaling pathways

The triple-negative breast cancer (TNBC) that comprises approximately 10%–20% of breast cancers is an aggressive subtype lacking effective therapeutics. Among various signaling pathways, mTORC1 and purinergic signals have emerged as potentially fruitful targets for clinical therapy of TNBC. Unfortunately, drugs targeting these signaling pathways do not successfully inhibit the progression of TNBC, partially due to the fact that these signaling pathways are essential for the function of all types of cells. In this study, we report that TRPML1 is specifically upregulated in TNBCs and that its genetic downregulation and pharmacological inhibition suppress the growth of TNBC. Mechanistically, we demonstrate that TRPML1 regulates TNBC development, at least partially, through controlling mTORC1 activity and the release of lysosomal ATP. Because TRPML1 is specifically activated by cellular stresses found in tumor microenvironments, antagonists of TRPML1 could represent anticancer drugs with enhanced specificity and potency. Our findings are expected to have a major impact on drug targeting of TNBCs.     

Exploring Rak tyrosine kinase function in breast cancer

Protein tyrosine kinases have been implicated in the regulation of many cellular events such as cellular proliferation, differentiation, and development. Deregulation of protein tyrosine kinase activity has been shown to result in human cancer. The majority of the protein tyrosine kinases studied to date localize to the cell membrane, where they function as components of signal transduction pathways. However, small group of nuclear tyrosine kinases has been identified that includes Rak. Our recent investigations demonstrated that Rak functions as a potent tumor suppressor by regulating PTEN protein stability and function. Rak also effectively suppresses phenotypes associated with in vitro transformation in breast cancer cells and tumorigenicity in vivo. Moreover, depletion of Rak is sufficient to induce tumorigenicity in mammary epithelial cells. However, the mechanisms by which Rak and its substrates function in cancer remain largely unexplored, leaving many potential therapeutic targets yet undiscovered. Therefore, fully elucidating the biological functions of Rak may contribute to effective therapeutic approaches for Rak-defective cancers.     

RB1 and p53 at the crossroad of EMT and triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a heterogeneous disease that includes Basal-like and Claudin-low tumors. The Claudin-low tumors are enriched for features associated with epithelial-to-mesenchymal transition (EMT) and possibly for tumor initiating cells. Primary TNBCs respond relatively well to conventional chemotherapy; however, metastatic disease is virtually incurable. Thus, there is a great interest in identifying specific therapeutic targets for TNBC. The tumor suppressor RB1 is frequently lost in Basal-like breast cancer. To test for a causative role of RB1 gene loss in BC and for its effect on specific subtypes, we deleted mouse Rb in mammary stem/bipotent progenitor cells. This led to diverse mammary tumors including TNBC, with a subset of the latter containing p53 mutations and exhibiting features of Basal-like BC or EMT. Combined mutation of Rb and p53 in mammary stem/bipotent progenitors induced EMT type tumors. Here, we review our findings and those of others, which connect Rb and p53 to EMT in TNBC. Furthermore, we discuss how by understanding this circuit and its vulnerabilities, we may identify novel therapy for TNBC.     

Geminin Overexpression Promotes Imatinib Sensitive Breast Cancer: A Novel Treatment Approach for Aggressive Breast Cancers,Including a Subset of Triple Negative

Breast cancer is the second leading cause of cancer-related deaths in women. Triple negative breast cancer (TNBC) is an aggressive subtype that affects 10–25% mostly African American women. TNBC has the poorest prognosis of all subtypes with rapid progression leading to mortality in younger patients. So far, there is no targeted treatment for TNBC. To that end, here we show that c-Abl is one of several tyrosine kinases that phosphorylate and activate geminin’s ability to promote TNBC. Analysis of >800 breast tumor samples showed that geminin is overexpressed in ∼50% of all tumors. Although c-Abl is overexpressed in ∼90% of all tumors, it is only nuclear in geminin overexpressing tumors. In geminin-negative tumors, c-Abl is only cytoplasmic. Inhibiting c-Abl expression or activity (using imatinib or nilotinib) prevented geminin Y150 phosphorylation, inactivated the protein, and most importantly converted overexpressed geminin from an oncogene to an apoptosis inducer. In pre-clinical orthotopic breast tumor models, geminin-overexpressing cells developed aneuploid and invasive tumors, which were suppressed when c-Abl expression was blocked. Moreover, established geminin overexpressing orthotopic tumors regressed when treated with imatinib or nilotinib. Our studies support imatinib/nilotonib as a novel treatment option for patients with aggressive breast cancer (including a subset of TNBCs)-overexpressing geminin and nuclear c-Abl.     

Expression Profiling during Mammary Epithelial Cell Three-Dimensional Morphogenesis Identifies PTPRO as a Novel Regulator of Morphogenesis and ErbB2-Mediated Transformation

Identification of genes that are upregulated during mammary epithelial cell morphogenesis may reveal novel regulators of tumorigenesis. We have demonstrated that gene expression programs in mammary epithelial cells grown in monolayer cultures differ significantly from those in three-dimensional (3D) cultures. We identify a protein tyrosine phosphate, PTPRO, that was upregulated in mature MCF-10A mammary epithelial 3D structures but had low to undetectable levels in monolayer cultures. Downregulation of PTPRO by RNA interference inhibited proliferation arrest during morphogenesis. Low levels of PTPRO expression correlated with reduced survival for breast cancer patients, suggesting a tumor suppressor function. Furthermore, we showed that the receptor tyrosine kinase ErbB2/HER2 is a direct substrate of PTPRO and that loss of PTPRO increased ErbB2-induced cell proliferation and transformation, together with tyrosine phosphorylation of ErbB2. Moreover, in patients with ErbB2-positive breast tumors, low PTPRO expression correlated with poor clinical prognosis compared to ErbB2-positive patients with high levels of PTPRO. Thus, PTPRO is a novel regulator of ErbB2 signaling, a potential tumor suppressor, and a novel prognostic marker for patients with ErbB2-positive breast cancers. We have identified the protein tyrosine phosphatase PTPRO as a regulator of three-dimensional epithelial morphogenesis of mammary epithelial cells and as a regulator of ErbB2-mediated transformation. In addition, we demonstrated that ErbB2 is a direct substrate of PTPRO and that decreased expression of PTPRO predicts poor prognosis for ErbB2-positive breast cancer patients. Thus, our results identify PTPRO as a novel regulator of mammary epithelial transformation, a potential tumor suppressor, and a predictive biomarker for breast cancer.     

Oncogenic tyrosine kinases target Dok-1 for ubiquitin-mediated proteasomal degradation to promote cell transformation

Cellular transformation induced by oncogenic tyrosine kinases is a multistep process involving activation of growth-promoting signaling pathways and inactivation of suppressor molecules. Dok-1 is an adaptor protein that acts as a negative regulator of tyrosine kinase-initiated signaling and opposes oncogenic tyrosine kinase-mediated cell transformation. Findings that its loss facilitates transformation induced by oncogenic tyrosine kinases suggest that Dok-1 inactivation could constitute an intermediate step in oncogenesis driven by these oncoproteins. However, whether Dok-1 is subject to regulation by oncogenic tyrosine kinases remained unknown. In this study, we show that oncogenic tyrosine kinases, including p210(bcr-abl) and oncogenic forms of Src, downregulate Dok-1 by targeting it for degradation through the ubiquitin-proteasome pathway. This process is dependent on the tyrosine kinase activity of the oncoproteins and is mediated primarily by lysine-dependent polyubiquitination of Dok-1. Importantly, restoration of Dok-1 levels strongly suppresses transformation of cells expressing oncogenic tyrosine kinases, and this suppression is more pronounced in the context of a Dok-1 mutant that is largely refractory to oncogenic tyrosine kinase-induced degradation. Our findings suggest that proteasome-mediated downregulation of Dok-1 is a key mechanism by which oncogenic tyrosine kinases overcome the inhibitory effect of Dok-1 on cellular transformation and tumor progression.     

Nuclear import of PTPN18 inhibits breast cancer metastasis mediated by MVP and importin β2

Distant metastasis is the primary cause of breast cancer-associated death. The existing information, such as the precise molecular mechanisms and effective therapeutic strategies targeting metastasis, is insufficient to combat breast cancer. This study demonstrates that the protein tyrosine phosphatase PTPN18 is downregulated in metastatic breast cancer tissues and is associated with better metastasis-free survival. Ectopic expression of PTPN18 inhibits breast cancer cell metastasis. PTPN18 is translocated from the cytoplasm to the nucleus by MVP and importin β2 in breast cancer. Then, nuclear PTPN18 dephosphorylates ETS1 and promotes its degradation. Moreover, nuclear PTPN18 but not cytoplasmic PTPN18 suppresses transforming growth factor-β signaling and epithelial-to-mesenchymal transition by targeting ETS1. Our data highlight PTPN18 as a suppressor of breast cancer metastasis and provide an effective antimetastatic therapeutic strategy.Subject terms: Breast cancer, Epithelial-mesenchymal transition     

Discovery and SAR studies of novel 2-anilinopyrimidine-based selective inhibitors against triple-negative breast cancer cell line MDA-MB-468

Triple-negative breast cancers (TNBCs) are characterized as an invasive and intractable subtype of breast cancers. Overexpression of epidermal growth factor receptor (EGFR) has been considered to be an important target for TNBC therapy, but efficacies of EGFR inhibitors in clinical trials are elusive. In this study, novel series of 2-anilinopyrimidines were synthesized in an effort to identify selective inhibitors against an EGFR-overexpressing TNBC cell line. Biological evaluation demonstrated that compounds 21 and 38, with a 4-methylpiperidine group and a high ClogP value, exhibited good potency and selectivity for the TNBC cell line. This study has provided evidence to support further development of 2-anilinopyrimidine-based TNBC selective inhibitors and investigation of the targets of compounds 21 and 38.     

Targeting the EGFR/PCNA Signaling Suppresses Tumor Growth of Triple-Negative Breast Cancer Cells with Cell-Penetrating PCNA Peptides

Tyrosine 211 (Y211) phosphorylation of proliferation cell nuclear antigen (PCNA) coincides with pronounced cancer cell proliferation and correlates with poor survival of breast cancer patients. In epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant cells, both nuclear EGFR (nEGFR) expression and PCNA Y211 phosphorylation are increased. Moreover, the resistance to EGFR TKI is a major clinical problem in treating EGFR-overexpressing triple-negative breast cancer (TNBC). Thus, effective treatment to combat resistance is urgently needed. Here, we show that treatment of cell-penetrating PCNA peptide (CPPP) inhibits growth and induces apoptosis of human TNBC cells. The Y211F CPPP specifically targets EGFR and competes directly for PCNA tyrosine Y211 phosphorylation and prevents nEGFR from binding PCNA in vivo; it also suppresses tumor growth by sensitizing EGFR TKI resistant cells, which have enhanced nEGFR function and abrogated classical EGFR membrane signaling. Furthermore, we identify an active motif of CPPP, RFLNFF (RF6 CPPP), which is necessary and sufficient to inhibit TKI-resistant TNBC cell growth of orthotopic implanted tumor in mice. Finally, the activity of its synthetic retro-inverted derivative, D-RF6 CPPP, on an equimolar basis, is more potent than RF6 CPPP. Our study reveals a drug candidate with translational potential for the future development of safe and effective therapeutic for EGFR TKI resistance in TNBC.     

A Comprehensive Immunologic Portrait of Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a high-risk malignancy due to its high capacity for invasion and lack of targeted therapy. Immunotherapy continues to demonstrate efficacy in a variety of cancers, and thus may be a promising strategy for TNBC given the limited therapeutic options currently available for TNBC. In this study, we performed an exhaustive analysis of immunogenic signatures in TNBC based on 2 large-scale breast cancer (BC) genomic data. We compared enrichment levels of 26 immune cell activities and pathways among TNBC, non-TNBC, and normal tissue, and within TNBCs of different genotypic or phenotypic features. We found that almost all analyzed immune activities and pathways had significantly higher enrichment levels in TNBC than non-TNBC. Elevated enrichment of these immune activities and pathways was likely to be associated with better survival prognosis in TNBC. This study demonstrated that TNBC likely exhibits the strongest immunogenicity among BC subtypes, and thus warrants the immunotherapeutic option for TNBC.     

Tyrosine phosphatases as a superfamily of tumor suppressors in colorectal cancer

Phosphorylation and dephosphorylation processes catalyzed by numerous kinases and phosphorylases are essential for cell homeostasis and may lead to disturbances in a variety of vital cellular pathways, such as cell proliferation and differentiation, and thus to complex diseases including cancer. As over 80 % of all oncogenes encode protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs), which can reverse the effects of tyrosine kinases, are very important tumor suppressors. Alterations in tyrosine kinase and phosphatase genes including point mutations, changes in epigenetic regulation, as well as chromosomal aberrations involving regions critical to these genes, are frequently observed in a variety of cancers. Colorectal cancer (CRC) is one of the most common cancers in humans. CRCs occur in a familial (about 15 % of all cases), hereditary (about 5%) and sporadic (almost 75-80 %) form. As genetic-environmental interrelations play an important role in the susceptibility to sporadic forms of CRCs, many studies are focused on genetic alterations in such tumors. Mutational analysis of the tyrosine phosphatome in CRCs has identified somatic mutations in PTPRG, PTPRT, PTPN3, PTPN13 and PTPN14. The majority of these mutations result in a loss of protein function. Also, alterations in the expression of these genes, such as decreased expression of PTPRR, PTPRO, PTPRG and PTPRD, mediated by epigenetic mechanisms have been observed in a variety of tumors. Since cancer is a social and global problem, there will be a growing number of studies on alterations in the candidate cancer genes, including protein kinases and phosphatases, to determine the origin, biology and potential pathways for targeted anticancer therapy.     

The biological function of SHP2 in human disease

Tyrosyl phosphorylation participates in various pathological and physiological processes, which are regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). The Src homology- 2 domain containing phosphatase SHP2 (encoded by PTPN11) is an important phosphatase, which was found to be implicated in the regulation of genetic disease, development, metabolic, neurological, muscle, skeletal disease and cancer. Germline mutations in PTPN11 cause the Noonan Syndrome, LEOPARD syndrome and metachondromatosis. Somatic PTPN11 mutations occur in hematologic malignancies and in solid tumors. SHP2 is also an important component in oncogenic signaling pathways. It may play different roles in different stages and positions of human cancers. Whether SHP2 is an oncogene or cancer suppressor gene remains to be elucidated. Elucidation of the regulatory mechanisms of SHP2 in human disease will provide new insights into disease and new targets for therapy. Here, we summarized the structural basis and recent research progression on SHP2 in various human disease, including genetic and cancer diseases.     

Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated

To approach the transmembrane signaling pathway in the cell-to-cell adherens junctions (AJ), AJ-specific tyrosine phosphorylation was analyzed. When various types of rat adult tissues were pretreated with sodium orthovanadate, a potent inhibitor of tyrosine phosphatase, immunofluorescence microscopy showed that anti-phosphotyrosine polyclonal antibody specifically stained the undercoat of the cell-to-cell AJ. This indicates that the tyrosine kinase activity is elevated at the undercoat of the cell-to-cell AJ of adult tissues. To identify tyrosine kinases responsible for the high level of tyrosine phosphorylation at AJ, we have performed in vitro phosphorylation experiments with cell-to-cell AJ isolated from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41) and immunoblotting analyses with specific antibodies for tyrosine kinases. As a result, three proto-oncogenic tyrosine kinases of src family, c-yes, c-src, and lyn kinases, were identified as major tyrosine kinases in the cell-to-cell AJ of hepatocytes. Furthermore, it was immunofluorescently shown that at least two of these kinases, c-yes and c-src kinases, were enriched at the cell-to-cell AJ of various types of cells including hepatocytes. Based on these findings, it is concluded that, in various types of cells, specific proto-oncogenic tyrosine kinases of src-family (c-yes and c-src) are enriched to work as signal mediators in the cell-to-cell AJ where the level of tyrosine phosphorylation is elevated.