The Src homology phosphotyrosyl phosphatase 2 (SHP2) is an essential transducer of mitogenic and cell survival signaling in the epidermal growth factor receptor (EGFR) signaling pathway. However, the role of SHP2 in aberrant EGFR and human EGFR2 (HER2) signaling and cancer, particularly in breast cancer, has not been investigated. Here, we report that SHP2 is required for mitogenic and cell survival signaling and for sustaining the transformation phenotypes of breast cancer cell lines that overexpress EGFR and HER2. Inhibition of SHP2 suppressed EGF-induced activation of the Ras-ERK and the phosphatidylinositol 3 kinase-Akt signaling pathways, abolished anchorage-independent growth, induced epithelial cell morphology and led to reversion to a normal breast epithelial phenotype. Furthermore, inhibition of SHP2 led to upregulation of E-cadherin (epithelial marker) and downregulation of fibronectin and vimentin (mesenchymal markers). These results indicate that SHP2 promotes breast cancer cell phenotypes by positively modulating mitogenic and cell survival signaling, by suppressing E-cadherin expression which is known to play a tumor suppressor role and by sustaining the mesenchymal state as evidenced by the positive impact on fibronectin and vimentin expression. Therefore, SHP2 promotes epithelial to mesenchymal transition, whereas its inhibition leads to mesenchymal to epithelial transition. On the basis of these premises, we propose that interference with SHP2 function might help treat breast cancer.
The Kruppel-like transcription factors (KLFs) 4 and 5 (KLF4/5) are coexpressed in mouse embryonic stem cells, where they function redundantly to maintain pluripotency. In mammary carcinoma, KLF4/5 can each impact the malignant phenotype, but potential linkages to drug resistance remain unclear. In primary human breast cancers, we observed a positive correlation between KLF4/5 transcript abundance, particularly in the human epidermal growth factor receptor 2 (HER2)-enriched subtype. Furthermore, KLF4/5 protein was rapidly upregulated in human breast cancer cells following treatment with the HER2/epidermal growth factor receptor inhibitor, lapatinib. In addition, we observed a positive correlation between these factors in the primary tumors of genetically engineered mouse models (GEMMs). In particular, the levels of both factors were enriched in the basal-like tumors of the C3(1) TAg (SV40 large T antigen transgenic mice under control of the C3(1)/prostatein promoter) GEMM. Using tumor cells derived from this model as well as human breast cancer cells, suppression of KLF4 and/or KLF5 sensitized HER2-overexpressing cells to lapatinib. Indicating cooperativity, greater effects were observed when both genes were depleted. KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL). Moreover, MCL1 was upregulated by lapatinib in a KLF4/5-dependent manner, and enforced expression of MCL1 in KLF4/5-deficient cells restored drug resistance. In addition, combined suppression of KLF4/5 in cultured tumor cells additively inhibited anchorage-independent growth, resistance to anoikis and tumor formation in immunocompromised mice. Consistent with their cooperative role in drug resistance and other malignant properties, KLF4/5 levels selectively stratified human HER2-enriched breast cancer by distant metastasis-free survival. These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies.
Background: Of all breast cancer subtypes, the basal-type/triple-negative breast cancer (BTBC) has the worst clinical outcome. The major reasons are the highly invasive nature of the disease and the limited treatment options available to patients. As opposed to the HER2−positive and the hormone-positive breast cancers, targeted therapy against BTBC is unavailable. These premises underscore the need for discovering and characterizing drug targets in BTBC. In this study, we have focused on the Src homology phosphotyrosyl phosphatase 2 (SHP) which seems to play pivotal roles in BTBC. The major reason for focusing on SHP2 is that it is an essential downstream effector of mitogenic and cell survival signaling downstream of receptor tyrosine kinases such as EGFR (HER1) and IGF-1R, and the cytoplasmic tyrosine kinase Src, which are known to be elevated in BTBC. Materials and Methods: The state of SHP2 and EGFR expression in BTBC tumors was determined by immunohistochemistry. The functional significance of SHP2 in BTBC cells was investigated by ablating its expression with specific shRNA and then assessing impact on mitogenic and cell survival signaling using immunoblotting with phospho-specific antibodies, effect on transformation using 3D cultures such as growth in soft agar and matrigel, consequence on cell motility and invasiveness using florescent-tagged matrigel. Furthermore, the impact of SHP2 inhibition on tumorigenesis was tested by intramammary transplantation in vivo and monitoring and analyzing tumor growth and metastasis. Results: We have shown that the SHP2 protein is elevated in BTBC tumors. More importantly, the elevated expression of SHP2 is highly correlated with overexpression of the EGFR (HER1), suggesting their potential synergistic role to promote BTBC. Inhibition of SHP2 in BTBC cells reversed transformation and suppressed proliferation, indicating their dependence on the function of SHP2. Moreover, inhibition of SHP2 abolished EGF-induced mitogenic and cell survival signaling, which is in agreement with its role in cell proliferation and transformation. More dramatic was that the invasive phenotype of BTBC cells in 3D matrigel was completely blocked by inhibition of SHP2, suggesting that the invasive property of these cells is dependent on SHP2. Even more dramatic was that the tumorigenic and metastatic potential of BTBC cells was abolished by SHP2 inhibition. Discussion: The current study demonstrates that SHP2 plays a pivotal role in promoting BTBC. Given that SHP2 is a tyrosine phosphatase with positive signaling role, its promotion of BTBC must occurs through promotion of tyrosine kinase signaling. Together, our results provide the first glimpse on the potential of SHP2 as a drug target in BTBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-15.
Background: The Src homology phosphotyrosyl phosphatase 2 (SHP2) acts as a transducer of mitogenic and cell survival signaling downstream of receptor tyrosine kinases, cytokine receptors and integrins. As such, SHP2 promotes cell growth, transformation, survival, and motility, among other processes. Recent studies suggest that SHP2 is overexpressed in breast cancer and is essential for the maintenance of the transformed phenotype both in the HER2−positive and the basal-type/triple-negative breast cancer (BTBC) cells. Materials and Methods: The expression of SHP2 in BTBC cells was suppressed constitutively by lenti virus-mediated transduction of specific shRNA. Impact of SHP2 inhibition on cell migration was determined by the monolayer wound healing assay. The SHP2 substrate-trapping mutant was used to identify FAK as an SHP2 substrate. Site-directed mutagenesis, binding studies and phosphatase assays were employed to further characterize FAK as a bona fide SHP2 substrate. Immunofluorescence microscopy was conducted to visualize the amounts of phosphorylated FAK and its location in the cell after adhesion. Results: We demonstrate that SHP2 is essential for the migration of BTBC cells as evidenced by loss of the enhanced migratory behavior upon its inhibition. Using the SHP2 substrate-trapping mutant, FAK was identified as a biological substrate of SHP2. Site-directed mutagenesis, binding studies and phosphatase assays confirmed that SHP2 indeed associates with and dephosphorylates FAK at the pY397 site, a site known to promote the focal adhesion activity of FAK. Immunofluorescence of breast cancer cells with SHP2 inhibition were found to have higher levels of FAK pY397 further confirming that FAK is an SHP2 substrate. The increase in pY397 level correlated with loss of cell polarity, enhanced focal adhesion formation, and acquisition of non-transformed morphology. Discussion: These results suggest that dephosphorylation of pY397 of FAK by SHP2 is the major mechanism by which it is able to modulate cancer cell migration. Together with previous findings, the current studies show that SHP2 is a suitable target for therapeutic intervention to combat breast cancer metastasis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-05.
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