Targeting SHP2 with an Active Site Inhibitor Blocks Signaling and Breast Cancer Cell Phenotypes.

The Src homology phosphotyrosyl phosphatase 2 (SHP2) is an oncogenic protein for which targeted therapies are being sought. In line with this idea, we have previously reported the development of a specific active site inhibitor named CNBDA that showed effectivity in suppressing the transformation phenotypes of breast cancer cells. To improve efficacy, we introduced limited modifications to the parent compound and tested potency in vitro and under cell culture conditions. Of these modifications, removal of one of the butyric acid groups led to the production of a compound named CNBCA, which showed a 5.7-fold better potency against the SHP2 enzyme activity in vitro. In addition, CNBCA showed better selectivity to SHP2 than the control PTPs (SHP1 and PTP1B) as determined by the phosphatase assay. Furthermore, CNBCA binds and inhibits enzyme activity of full-length SHP2 in cellular contexts, downregulates SHP2 mediated signaling, and suppresses breast cancer cell phenotypes, including cell proliferation, colony formation, and mammosphere growth. These findings show that targeting SHP2 with CNBCA is effective against the cancerous properties of breast cancer cells.

Design and synthesis of improved active-site SHP2 inhibitors with anti-breast cancer cell effects.

The Src homology containing phosphotyrosyl phosphatase 2 (SHP2) is a bona fide oncogene particularly in cancers driven by overexpression of receptor tyrosine kinases (RTKs). As such, there is a growing interest to target SHP2 in cancer. Based on these premises, several active site (type I) and allosteric site (type II) inhibitors have been developed, but no SHP2 targeting therapies have reached the clinic yet. In an effort to fill these gaps, we embarked on producing optimized versions of our parent active-site SHP2 inhibitor CNBDA. The objectives were to produce derivatives with increased inhibitory potential and improved selectivity. Accordingly, we designed derivatives around the CNBDA scaffold and predicted their binding property by in silico molecular modeling. Based on comparative differences in free energy of binding to the SHP2 versus the SHP1 active sites, ten were selected, chemically synthesized, and evaluated by NMR and mass spectroscopy for structural integrity. Among the ten derivatives, BPDA2 was found to be the most potent and highly selective compound, inhibiting the SHP2 enzyme activity with an IC50 of 92 nM when DiFMUP was used as a substrate and with an IC50 of 47 nM when pNPP was used as a substrate. Furthermore, enzyme kinetic analyses showed that BPDA2 is a competitive SHP2 inhibitor. Selectivity comparisons in a PTPase assay using DiFMUP as a substrate demonstrated that BPDA2 is more selective to SHP2 than to SHP1 and PTP1B by more than 369-fold and 442-fold, respectively. Evaluation with a cellular thermal shift assay (CETSA) confirmed that BPDA2 binds to wild-type SHP2 in a cellular context, and stabilizes it in solution. Treatment of cells with DBDA2 downregulates mitogenic and cell survival signaling and RTK expression in a concentration dependent manner. Furthermore, treatment of cells with BPDA2 suppresses anchorage independent growth and cancer stem cell properties of breast cancer cells. Overall, data described in this report show that BPDA2 is a more potent derivative of CNBDA with a highly improved selectivity for SHP2.

SHP2 Potentiates the Oncogenic Activity of ß-Catenin to Promote Triple-Negative Breast Cancer.

Previous studies have reported dysregulated cytoplasmic and nuclear expression of the ß-catenin protein in triple-negative breast cancer (TNBC) in the absence of Wnt signaling pathway dysregulation. However, the mechanism that sustains ß-catenin protein dysregulation independent of Wnt signaling is not understood. In this study, we show that Src homology phosphotyrosyl phosphatase 2 (SHP2) is essential for ß-catenin protein stability and for sustaining the cytoplasmic and nuclear pools in TNBC cells. The first evidence for this possibility came from immunofluorescence (IF) and immunoblotting (IB) studies that showed that inhibition of SHP2 induces E-cadherin expression and depletion of cytoplasmic and nuclear ß-catenin, which in turn confers adherence junction mediated cell-cell adhesion. We further show that SHP2 promotes ß-catenin protein stability by mediating the inactivation of GSK3ß through its positive effect on Akt and ERK1/2 activation, which was confirmed by direct pharmacologic inhibition of the PI3K-Akt and the MEK-ERK signaling pathway. Finally, we show that SHP2-stabilized ß-catenin contributes to TNBC cell growth, transformation, cancer stem cell (CSC) properties, and tumorigenesis and metastasis. Overall, the findings in this report show that SHP2 mediates ß-catenin protein stability to promote TNBC. IMPLICATIONS: Data presented in this article demonstrates that SHP2 positively regulates ß-catenin protein stability, which in turn promotes triple-negative breast cancer (TNBC) cell transformation, tumorigenesis, and metastasis.

Novel Small-Molecule Inhibitor for the Oncogenic Tyrosine Phosphatase SHP2 with Anti-Breast Cancer Cell Effects.

The oncogenic property of the Src homology phosphotyrosine phosphatase 2 (SHP2) is well-known, but developing specific inhibitors has been very difficult. Based on our previous reports that showed the importance of acidic residues surrounding SHP2 substrate phosphotyrosines for specific recognition, we have rationally designed and chemically synthesized a small-molecule SHP2 inhibitor named 4,4'-(4'-carboxy)-4-nonyloxy-[1,1'-biphenyl]-3,5-diyl)dibutanoic acid (CNBDA). Molecular modeling predicted that CNBDA packs well into the SHP2 active site and makes extended interactions primarily with positively charged and polar amino acids surrounding the active site. In vitro PTPase assays showed that CNBDA inhibits SHP2 with an IC50 of 5 µM. However, the IC50 of CNBDA toward SHP1, the close structural homologue of SHP2, was 125 µM, suggesting an approximately 25-fold effectiveness against SHP2 than SHP1. Because SHP2 is known for its positive role in breast cancer (BC) cell biology, we tested the effect of SHP2 inhibition with CNBDA in HER2-positive BC cells. Treatment with CNBDA suppressed cell proliferation in 2D culture, anchorage-independent growth in soft agar, and mammosphere (tumorisphere) formation in suspension cultures in a concentration-dependent manner. Furthermore, CNBDA inhibited EGF-induced signaling and expression of HER2 by inhibiting the PTPase activity of SHP2 in BC cells. These findings suggest that CNBDA is a promising anti-SHP2 lead compound with anti-BC cell effects.

A specific amino acid context in EGFR and HER2 phosphorylation sites enables selective binding to the active site of Src homology phosphatase 2 (SHP2).

The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1-4 or the human homologs HER1-4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr992-EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity in vitro and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors.

Conditional knockout of SHP2 in ErbB2 transgenic mice or inhibition in HER2-amplified breast cancer cell lines blocks oncogene expression and tumorigenesis.

Overexpression of the human epidermal growth factor receptor 2 (HER2) is the cause of HER2-positive breast cancer (BC). Although HER2-inactivating therapies have benefited BC patients, development of resistance and disease recurrence have been the major clinical problems, pointing to a need for alternative therapeutic strategies. For that to happen, proteins that play critical roles in the biology of HER2-induced tumorigenesis have to be identified and characterized. Here, we show that the Src homology phosphotyrosyl phosphatase 2 (Shp2) encoded by the Ptpn11 gene is a requisite for ErbB2-induced tumorigenesis. We report that conditional knockout of Shp2 alleles in the ErbB2 BC model mice abrogates mammary tumorigenesis by blocking the expression of the ErbB2 transgene. We also show that inhibition of SHP2 encoded by the PTPN11 gene in the HER2-amplified BC cells induces a normal-like cellular phenotype and suppresses tumorigenesis and metastasis by blocking HER2 overexpression. These findings demonstrate that ErbB2-induced tumors in mice or xenograft tumors induced by transplantation of HER2-amplified BC cells are vulnerable to SHP2 inhibition since it abrogates the expression of the very oncogene that causes of the disease. This report paves the way for developing SHP2-targeting therapies for BC treatment in the future.

SHP2 acts both upstream and downstream of multiple receptor tyrosine kinases to promote basal-like and triple-negative breast cancer.

INTRODUCTION: Dysregulated receptor tyrosine kinase (RTK) signaling is a common occurrence in basal-like and triple-negative breast cancer (BTBC). As a result, RTK-targeting therapies have been initiated but proved difficult, mainly owing to the multiplicity of dysregulated RTKs. Hence, targeting master regulators of RTK signaling might alleviate this obstacle. Before that, however, defining the mechanism of such molecules is required. In this report, we show that the Src homology phosphotyrosyl phosphatase 2 (SHP2) is a master regulator of RTK expression and signaling in BTBC. METHODS: Xenograft tumor growth studies were used to determine the effect of SHP2 inhibition on tumorigenesis and/or metastasis. Cell proliferation rate, anchorage-independent growth, mammosphere formation, and ALDEFLUOR assays were used to compare the relative functional importance of SHP2 and the epidermal growth factor receptor (EGFR) in BTBC cells. Immunohistochemistry and immunofluorescence analyses were used to determine the state of SHP2 and EGFR coexpression in BTBC. Analysis of mitogenic and cell survival signaling was performed to show SHP2's role in signaling by multiple RTKs. RESULTS: Inhibition of SHP2 in BTBC cells suppresses their tumorigenic and metastatic properties. Because EGFR is the most commonly dysregulated RTK in BTBC, we first tested the effect of SHP2 inhibition on EGFR signaling and found that SHP2 is important not only for mediation of the Ras/extracellular signal-regulated kinase and the phosphatidyl inositol 3-kinase/Akt signaling pathways but also for the expression of the receptor itself. The existence of a tight association between SHP2 and EGFR expression in tumors and cell lines further suggested the importance of SHP2 in EGFR expression. Comparison of relative biological significance showed the superiority of SHP2 inhibition over that of EGFR, suggesting the existence of additional RTKs regulated by SHP2. Indeed, we found that the expression as well as the signaling efficiency of c-Met and fibroblast growth factor receptor 1, two other RTKs known to be dysregulated in BTBC, are SHP2-dependent. To our knowledge, this is the first demonstration of SHP2 acting both upstream and downstream of RTKs to promote signaling. CONCLUSIONS: SHP2 upregulates the expression and signaling of multiple RTKs to promote BTBC. These findings provide a mechanistic explanation for the superiority of SHP2 inhibition in BTBC.

Inhibition of SHP2 in basal-like and triple-negative breast cells induces basal-to-luminal transition, hormone dependency, and sensitivity to anti-hormone treatment.

BACKGROUND: The Src homology phosphotyrosyl phosphatase 2 (SHP2) is a positive effector of cell growth and survival signaling as well transformation induced by multiple tyrosine kinase oncogenes. Since the basal-like and triple-negative breast cancer (BTBC) is characterized by dysregulation of multiple tyrosine kinase oncogenes, we wanted to determine the importance of SHP2 in BTBC cell lines. METHODS: Short hairpin RNA-based and dominant-negative expression-based SHP2 inhibition techniques were used to interrogate the functional importance of SHP2 in BTBC cell biology. In addition, cell viability and proliferation assays were used to determine hormone dependency for growth and sensitivity to anti-estrogen treatment. RESULTS: We show that inhibition of SHP2 in BTBC cells induces luminal-like epithelial morphology while suppressing the mesenchymal and invasive property. We have termed this process as basal-to-luminal transition (BLT). The occurrence of BLT was confirmed by the loss of the basal marker alpha smooth muscle actin and the acquisition of the luminal marker cytokeratin 18 (CK18) expression. Furthermore, the occurrence of BLT led to estrogen receptor alpha (ERα) expression, hormone dependency, and sensitivity to tamoxifen treatment. CONCLUSIONS: Our data show that inhibition of SHP2 induces BLT, ERα expression, dependency on estrogen for growth, and sensitivity to anti-hormone therapy. Therefore, inhibition of SHP2 may provide a therapeutic benefit in basal-like and triple-negative breast cancer.

The tyrosine phosphatase SHP2 regulates focal adhesion kinase to promote EGF-induced lamellipodia persistence and cell migration.

The Src homology phosphotyrosyl phosphatase 2 (SHP2) is a positive effector of receptor tyrosine kinases (RTK) signaling. Furthermore, SHP2 is known to promote cell migration and invasiveness, key steps in cancer metastasis. To date, however, the mechanism by which SHP2 regulates cell movement is not fully understood. In the current report, a new role for SHP2 in regulating cell migration has been suggested. We show that SHP2 mediates lamellipodia persistence and cell polarity to promote directional cell migration in the MDA-MB231 and the MDA-MB468 basal-like and triple-negative breast cancer cell lines. We further show that SHP2 modulates the activity of focal adhesion kinase (FAK) by dephosphorylating pTyr397, the autophosphorylation site that primes FAK function. Because hyperactivation of FAK is known to counter the maturation of nascent focal complexes to focal adhesions, we propose that one of the mechanisms by which SHP2 promotes lamellipodia persistence is by downregulating FAK activity through dephosphorylation of pTyr397. The finding that inhibition of FAK activity partially restores EGF-induced lamellipodia persistence and cell migration in SHP2-silenced cells supports our proposition that SHP2 promotes growth factor-induced cell movement by acting, at least in part, on FAK. However, the effect of SHP2 inhibition in nonstimulated cells seems FAK independent as there was no significant difference between the control and the SHP2-silenced cells in pY397-FAK levels. Also, FAK inhibition did not rescue Golgi orientation defects in SHP2-silenced cells, suggesting that SHP2 acts through other mechanisms to promote cell polarity.

The signaling and transformation potency of the overexpressed HER2 protein is dependent on the normally-expressed EGFR.

Human epidermal growth factor receptor 2 (HER2) belongs to the EGFR family of receptor tyrosine kinases that comprises four members. As opposed to the other family members, HER2 does not require ligand binding for activation. Hence, HER2 molecules can undergo spontaneous dimerization, autophosphorylation and activation of downstream signaling pathways especially under conditions of overexpression, a commonly encountered phenomenon in breast cancer. In this study, we sought to investigate the mechanism by which HER2 musters signaling and transformation potency. We show that HER2 overexpression per se induces a significant increase in basal mitogenic and cell survival signaling, which was augmented by EGF stimulation. Inhibition of the normally expressed EGFR significantly suppressed the ability of overexpressed HER2 to induce enhanced signaling and cell transformation, suggesting that HER2 requires the EGFR and potentially other members to maximize its signaling and transformation potency. The novel observation revealed by prolonged EGF stimulation studies was the biphasic signaling pattern in the presence of HER2 overexpression that suggested the induction of a short-circuited mechanism, permitting sustained signaling. Our results further show that the short-circuited signaling was due to the re-shuttling of internalized receptor molecules to the Rab11-positive recycling endosomes, while suppressing channeling to the LAMP1-positive lysosome-targeting endosomes. Therefore, HER2's oncogenicity is dependent, not only on its constitutively active nature, but also on its ability to muster collaborative signaling from family members through modulation of ligand-induced receptor regulation.

Molecular mechanism for SHP2 in promoting HER2-induced signaling and transformation.

The Src homology phosphotyrosyl phosphatase 2 (SHP2) plays a positive role in HER2-induced signaling and transformation, but its mechanism of action is poorly understood. Given the significance of HER2 in breast cancer, defining a mechanism for SHP2 in the HER2 signaling pathway is of paramount importance. In the current report we show that SHP2 positively modulates the Ras-extracellular signal-regulated kinase 1 and 2 and the phospoinositide-3-kinase-Akt pathways downstream of HER2 by increasing the half-life the activated form of Ras. This is accomplished by dephosphorylating an autophosphorylation site on HER2 that serves as a docking platform for the SH2 domains of the Ras GTPase-activating protein (RasGAP). The net effect is an increase in the intensity and duration of GTP-Ras levels with the overall impact of enhanced HER2 signaling and cell transformation. In conformity to these findings, the HER2 mutant that lacks the SHP2 target site exhibits an enhanced signaling and cell transformation potential. Therefore, SHP2 promotes HER2-induced signaling and transformation at least in part by dephosphorylating a negative regulatory autophosphorylation site. These results suggest that SHP2 might serve as a therapeutic target against breast cancer and other cancers characterized by HER2 overexpression.

Mutation of Thr466 in SHP2 abolishes its phosphatase activity, but provides a new substrate-trapping mutant.

Most classical phosphotyrosyl phosphatases (PTPs), including the Src homology phosphotyrosyl phosphatase 2 (SHP2) possess a Thr or a Ser residue immediately C-terminal to the invariant Arg in the active site consensus motif (H/V-C-X5-R-S/T), also known as the "signature motif". SHP2 has a Thr (Thr466) at this position, but its importance in catalysis has not been investigated. By employing site-directed mutagenesis, phosphatase assays and substrate-trapping studies, we demonstrate that Thr466 is critical for the catalytic activity of SHP2. Its mutation to Ala abolishes phosphatase activity, but provides a new substrate-trapping mutant. We further show that the nucleophilic Cys459 is not involved in substrate trapping by Thr466Ala-SHP2 (T/A-SHP2). Mutation of Thr466 does not cause significant structural changes in the active site as revealed by the trapping of the epidermal growth factor receptor (EGFR), the physiological substrate of SHP2, and by orthovanadate competition experiments. Based on these results and previous other works, we propose that the role of Thr466 in the catalytic process of SHP2 could be stabilizing the sulfhydryl group of Cys459 in its reduced state, a state that enables nucleophilic attack on the phosphate moiety of the substrate. The T/A-SHP2 harbors a single mutation and specifically interacts with the EGFR. Since the nucleophilic Cys459 and the proton donor Asp425 are intact in the T/A-SAHP2, it offers an excellent starting material for solving the structure of SHP2 in complex with its physiological substrate.

The phosphotyrosine phosphatase SHP2 is a critical mediator of transformation induced by the oncogenic fibroblast growth factor receptor 3.

Receptor tyrosine kinases (RTKs) such as the fibroblast growth factor receptor (FGFR) and the epidermal growth factor receptor are overexpressed in a variety of cancers. In addition to overexpression, the FGFRs are found mutated in some cancers. The Src homology 2 domain-containing phosphotyrosine phosphatase (SHP2) is a critical mediator of RTK signaling, but its role in oncogenic RTK-induced cell transformation and cancer development is largely unknown. In the current report, we demonstrate that constitutively activated FGFR3 (K/E-FR3) transforms NIH-3T3 cells, and that SHP2 is a critical mediator of this transformation. Infection of K/E-FR3-transformed 3T3 cells with a retrovirus carrying a dominant-negative mutant of SHP2 (C/S-SHP2) retarded cell growth, reversed the transformation phenotype and inhibited focus-forming ability. Furthermore, treatment of K/E-FR3-transformed NIH-3T3 cells with PD98059 or LY294002, specific inhibitors of MEK and PI3K, respectively, inhibited focus formation. Biochemical analysis showed that K/E-FR3 activates the Ras-ERK and the PI3K signaling pathways, and that the C/S SHP2 mutant suppressed this effect via competitive displacement of interaction of the endogenous SHP2 with FRS2. However, the C/S SHP2 protein did not show any effect on receptor autophosphorylation, FRS2 tyrosine phosphorylation or interaction of Grb2 with K/E-FR3 or FRS2. Together, the results show that K/E-FR3 is transforming and that the Ras-ERK and the PI3K-Akt signaling pathways, which are positively regulated by SHP2, are important for K/E-FR3-induced transformation.

Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling.

The Src homology 2-containing phosphotyrosine phosphatase (SHP2) is primarily a positive effector of receptor tyrosine kinase signaling. However, the molecular mechanism by which SHP2 effects its biological function is unknown. In this report, we provide evidence that defines the molecular mechanism and site of action of SHP2 in the epidermal growth factor-induced mitogenic pathway. We demonstrate that SHP2 acts upstream of Ras and functions by increasing the half-life of activated Ras (GTP-Ras) in the cell by interfering with the process of Ras inactivation catalyzed by Ras GTPase-activating protein (RasGAP). It does so by inhibition of tyrosine phosphorylation-dependent translocation of RasGAP to the plasma membrane, to its substrate (GTP-Ras) microdomain. Inhibition is achieved through the dephosphorylation of RasGAP binding sites at the level of the plasma membrane. We have identified Tyr992 of the epidermal growth factor receptor (EGFR) to be one such site, since its mutation to Phe renders the EGFR refractory to the effect of dominant-negative SHP2. To our knowledge, this is the first report to outline the site and molecular mechanism of action of SHP2 in EGFR signaling, which may also serve as a model to describe its role in other receptor tyrosine kinase signaling pathways.

Development of an efficient "substrate-trapping" mutant of Src homology phosphotyrosine phosphatase 2 and identification of the epidermal growth factor receptor, Gab1, and three other proteins as target substrates.

Src homology containing phosphotyrosine phosphatase 2 (SHP2) is a positive effector of growth factor, cytokine, and integrin signaling. However, neither its physiological substrate nor its mechanism of action in tyrosine kinase signaling has been demonstrated. We reasoned that the identification of physiological substrates of SHP2 would be a stepping stone in elucidating its mechanism of action, and, thus, we constructed a potent trapping mutant of SHP2. Surprisingly, the frequently used Asp to Ala substitution did not give rise to a trapping mutant. However, we were able to develop an efficient trapping mutant of SHP2 by introducing Asp to Ala and Cys to Ser double mutations. The double mutant (DM) protein identified the epidermal growth factor receptor (EGFR), the Grb2 binder 1, and three other, as yet unidentified, phosphotyrosyl proteins as candidate physiological substrates. Given that substrate trapping occurred in intact cells and that the interaction was very specific, it is highly likely that EGFR and Gab1 represent physiological SHP2 substrates. Therefore, the DM protein would serve as an important tool in future SHP2 studies, including identification of p190, p150, and p90.