FAK activation is required for IGF1R-mediated regulation of EMT, migration, and invasion in mesenchymal triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is a highly metastatic disease that currently lacks effective prevention and treatment strategies. The insulin-like growth factor 1 receptor (IGF1R) and focal adhesion kinase (FAK) signaling pathways function in numerous developmental processes, and alterations in both are linked with a number of common pathological diseases. Overexpression of IGF1R and FAK are closely associated with metastatic breast tumors. The present study investigated the interrelationship between IGF1R and FAK signaling in regulating the malignant properties of TNBC cells. Using small hairpin RNA (shRNA)-mediated IGF1R silencing methods, we showed that IGF1R is essential for sustaining mesenchymal morphologies of TNBC cells and modulates the expression of EMT-related markers. We further showed that IGF1R overexpression promotes migratory and invasive behaviors of TNBC cell lines. Most importantly, IGF1R-driven migration and invasion is predominantly mediated by FAK activation and can be suppressed using pharmacological inhibitors of FAK. Our findings in TNBC cells demonstrate a novel role of the IGF1R/FAK signaling pathway in regulating critical processes involved in the metastatic cascade. These results may improve the current understanding of the basic molecular mechanisms of TNBC metastasis and provide a strong rationale for co-targeting of IGF1R and FAK as therapy for mesenchymal TNBCs.

Insulin-like growth factor-1 receptor signaling increases the invasive potential of human epidermal growth factor receptor 2-overexpressing breast cancer cells via Src-focal adhesion kinase and forkhead box protein M1.

Resistance to the human epidermal growth factor receptor (HER2)-targeted antibody trastuzumab is a major clinical concern in the treatment of HER2-positive metastatic breast cancer. Increased expression or signaling from the insulin-like growth factor-1 receptor (IGF-1R) has been reported to be associated with trastuzumab resistance. However, the specific molecular and biologic mechanisms through which IGF-1R promotes resistance or disease progression remain poorly defined. In this study, we found that the major biologic effect promoted by IGF-1R was invasion, which was mediated by both Src-focal adhesion kinase (FAK) signaling and Forkhead box protein M1 (FoxM1). Cotargeting IGF-1R and HER2 using either IGF-1R antibodies or IGF-1R short hairpin RNA in combination with trastuzumab resulted in significant but modest growth inhibition. Reduced invasion was the most significant biologic effect achieved by cotargeting IGF-1R and HER2 in trastuzumab-resistant cells. Constitutively active Src blocked the anti-invasive effect of IGF-1R/HER2 cotargeted therapy. Furthermore, knockdown of FoxM1 blocked IGF-1-mediated invasion, and dual targeting of IGF-1R and HER2 reduced expression of FoxM1. Re-expression of FoxM1 restored the invasive potential of IGF-1R knockdown cells treated with trastuzumab. Overall, our results strongly indicate that therapeutic combinations that cotarget IGF-1R and HER2 may reduce the invasive potential of cancer cells that are resistant to trastuzumab through mechanisms that depend in part on Src and FoxM1.

Heat shock protein 90 promotes epithelial to mesenchymal transition, invasion, and migration in colorectal cancer.

Epithelial to mesenchymal transition (EMT), invasion, and motility are essential steps in colorectal cancer (CRC) metastasis regulated by HIF-1α and NF-κB. Since HSP90 activates HIF-1α and NF-κB, we hypothesized that inhibition of HSP90 leads to inhibition of HIF-1α and NF-κB resulting in inhibition of EMT, invasion, and motility. Treatment of colorectal cancer cell lines HT-29 and HCT-116 with ganetespib at 50 nM for 24 h inhibited EMT (downregulated vimentin and upregulated E-cadherin), matrigel invasion, and spheroid migration. Ganetespib treatment or HSP90 knockdown downregulated molecular pathways associated with EMT, invasion, and motility. The overexpression of HIF-1α or NF-κB resulted in increased EMT, invasion, and motility in both cell lines and these effects were inhibited by ganetespib. Similar effects were observed in animal xenografts treated with ganetespib. Taken together, our data demonstrate for the first time that inhibition of HSP90 downregulates both HIF-1α and NF-κB leading to inhibition of EMT, motility, and invasiveness in colorectal cancer.

Snail promotes epithelial mesenchymal transition in breast cancer cells in part via activation of nuclear ERK2.

Snail transcription factor is up-regulated in several cancers and associated with increased tumor migration and invasion via induction of epithelial-to-mesenchymal transition (EMT). MAPK (ERK1/2) signaling regulates cellular processes including cell motility, adhesion, and invasion. We investigated the regulation of ERK1/2 by Snail in breast cancer cells. ERK1/2 activity (p-ERK) was higher in breast cancer patient tissue as compared to normal tissue. Snail and p-ERK were increased in several breast cancer cell lines as compared to normal mammary epithelial cells. Snail knockdown in MDA-MB-231 and T47-D breast cancer cells decreased or re-localized p-ERK from the nuclear compartment to the cytoplasm. Snail overexpression in MCF-7 breast cancer cells induced EMT, increased cell migration, decreased cell adhesion and also increased tumorigenicity. Snail induced nuclear translocation of p-ERK, and the activation of its subcellular downstream effector, Elk-1. Inhibiting MAPK activity with UO126 or knockdown of ERK2 isoform with siRNA in MCF-7 Snail cells reverted EMT induced by Snail as shown by decreased Snail and vimentin expression, decreased cell migration and increased cell adhesion. Overall, our data suggest that ERK2 isoform activation by Snail in aggressive breast cancer cells leads to EMT associated with increased cell migration and decreased cell adhesion. This regulation is enhanced by positive feedback regulation of Snail by ERK2. Therefore, therapeutic targeting of ERK2 isoform may be beneficial for breast cancer.

Antiangiogenic effects of ganetespib in colorectal cancer mediated through inhibition of HIF-1α and STAT-3.

Hypoxia-inducible factors (HIFs) and STAT-3 play essential roles in angiogenesis. HIF-1α and STAT-3 are clients of the heat shock protein 90 (HSP90). We hypothesized that ganetespib, a potent HSP90 inhibitor, would disrupt angiogenesis in colorectal cancer (CRC) through inhibition of HIF-1α and STAT-3. CRC cell lines (HCT116 and HT29) were used in all the experiments. Egg CAM and HUVEC assays revealed decreased angiogenesis in ganetespib treated cell lines. Ganetespib inhibited matrigel plug vascularization and tumor growth of xenografts. Significant inhibition of PDGFA, FGF2, Ang-1, Ang-2, TGFß1, VEGF, HIF-1α and STAT-3 expression was observed in both cell lines treated ganetespib. HIF-1α overexpression resulted in the increase VEGF and STAT-3 expression and this was inhibited by ganetespib. HIF-1α knockdown inhibited VEGF and STAT-3 expression. STAT-3 knockdown inhibited VEGF but not HIF-1α expression. HSP90, STAT-3 and VEGF expression was significantly higher in CRC compared to adjacent normal tissue. Significant downregulation of PDGFA, FGF2, Ang-1, Ang-2, TGFß1, VEGF, STAT-3 and HIF-1α mRNA was observed in the post ganetespib treatment tumor samples from patients with rectal cancer. These results collectively suggest that inhibition of HSP90 is a promising antiangiogenic strategy in CRC. HSP90 angiogenic effects are mediated through HIF-1α and STAT-3.

Integral role of PTP1B in adiponectin-mediated inhibition of oncogenic actions of leptin in breast carcinogenesis.

The molecular effects of obesity are mediated by alterations in the levels of adipocytokines. High leptin level associated with obese state is a major cause of breast cancer progression and metastasis, whereas adiponectin is considered a "guardian angel adipocytokine" for its protective role against various obesity-related pathogenesis including breast cancer. In the present study, investigating the role of adiponectin as a potential inhibitor of leptin, we show that adiponectin treatment inhibits leptin-induced clonogenicity and anchorage-independent growth. Leptin-stimulated migration and invasion of breast cancer cells is also effectively inhibited by adiponectin. Analyses of the underlying molecular mechanisms reveal that adiponectin suppresses activation of two canonical signaling molecules of leptin signaling axis: extracellular signal-regulated kinase (ERK) and Akt. Pretreatment of breast cancer cells with adiponectin protects against leptin-induced activation of ERK and Akt. Adiponectin increases expression and activity of the physiological inhibitor of leptin signaling, protein tyrosine phosphatase 1B (PTP1B), which is found to be integral to leptin-antagonist function of adiponectin. Inhibition of PTP1B blocks adiponectin-mediated inhibition of leptin-induced breast cancer growth. Our in vivo studies show that adenovirus-mediated adiponectin treatment substantially reduces leptin-induced mammary tumorigenesis in nude mice. Exploring therapeutic strategies, we demonstrate that treatment of breast cancer cells with rosiglitazone results in increased adiponectin expression and inhibition of migration and invasion. Rosiglitazone treatment also inhibits leptin-induced growth of breast cancer cells. Taken together, these data show that adiponectin treatment can inhibit the oncogenic actions of leptin through blocking its downstream signaling molecules and raising adiponectin levels could be a rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels.

Combinatorial effects of lapatinib and rapamycin in triple-negative breast cancer cells.

Triple-negative breast cancers, which lack estrogen receptor, progesterone receptor, and HER2/neu overexpression, account for approximately 15% of breast cancers, but occur more commonly in African Americans. The poor survival outcomes seen with triple-negative breast cancers patients are, in part, due to a lack of therapeutic targets. Epidermal growth factor receptor (EGFR) is overexpressed in 50% of triple-negative breast cancers, but EGFR inhibitors have not been effective in patients with metastatic breast cancers. However, mTOR inhibition has been shown to reverse resistance to EGFR inhibitors. We examined the combination effects of mTOR inhibition with EGFR inhibition in triple-negative breast cancer in vitro and in vivo. The combination of EGFR inhibition by using lapatinib and mTOR inhibition with rapamycin resulted in significantly greater cytotoxicity than the single agents alone and these effects were synergistic in vitro. The combination of rapamycin and lapatinib significantly decreased growth of triple-negative breast cancers in vivo compared with either agent alone. EGFR inhibition abrogated the expression of rapamycin-induced activated Akt in triple-negative breast cancer cells in vitro. The combination of EGFR and mTOR inhibition resulted in increased apoptosis in some, but not all, triple-negative cell lines, and these apoptotic effects correlated with a decrease in activated eukaryotic translation initiation factor (eIF4E). These results suggest that mTOR inhibitors could sensitize a subset of triple-negative breast cancers to EGFR inhibitors. Given the paucity of effective targeted agents in triple-negative breast cancers, these results warrant further evaluation.

Reciprocal regulation of ZEB1 and AR in triple negative breast cancer cells.

Zinc-finger enhancer binding protein (ZEB1) is a transcription factor involved in the progression of cancer primarily through promoting epithelial to mesenchymal transition (EMT). ZEB1 represses the expression of E-cadherin by binding to E-box sequences in the promoter, thus decreasing epithelial differentiation. We show that ZEB1 and androgen receptor (AR) cross-talk in triple negative breast cancer cell lines. Chromatin immunoprecipitation analysis demonstrates that ZEB1 binds directly to the E-box located in the AR promoter. ZEB1 suppression by stably transfecting shRNA in a triple negative breast cancer cell line resulted in a decrease of AR mRNA, protein, and AR downstream targets. ZEB1 knockdown in triple negative breast cancer cells sensitized the cells to bicalutamide by reducing migration compared to the control cells. Conversely, blockade of AR signaling with bicalutamide resulted in a suppression of ZEB1 protein expression in two triple negative breast cancer cell lines. Furthermore, using a breast cancer tissue microarray, a majority of triple negative breast cancers exhibit positive staining for both ZEB1 and AR. Taken together, these results indicate that ZEB1 and AR regulate each other to promote cell migration in triple negative breast cancer cells.

Bidirectional crosstalk between leptin and insulin-like growth factor-I signaling promotes invasion and migration of breast cancer cells via transactivation of epidermal growth factor receptor.

Obesity is an independent risk factor for breast cancer, and obese breast cancer patients exhibit a higher risk for larger tumor burden and increased metastasis. Obesity, as associated with metabolic syndrome, results in an increase in circulating insulin-like growth factor (IGF), which acts as a mitogen. In addition, higher plasma level of adipocytokine leptin is associated with obesity. In the present study, we show that cotreatment with leptin and IGF-I significantly increases proliferation as well as invasion and migration of breast cancer cells. We found a novel bidirectional crosstalk between leptin and IGF-I signaling; IGF-I induced phosphorylation of leptin receptor (Ob-Rb) and leptin induced phosphorylation of IGF-I receptor (IGF-IR), whereas cotreatment induced synergistic phosphorylation and association of Ob-Rb and IGF-IR along with activation of downstream effectors, Akt and extracellular signal-regulated kinase. Leptin increased phosphorylation of IGF signaling molecules insulin-receptor substrate (IRS)-1 and IRS-2. Interestingly, we found that leptin and IGF-I cotreatment synergistically transactivated epidermal growth factor receptor (EGFR), depending on the proteolytic release of EGFR ligands, as the broad-spectrum matrix metalloproteinase inhibitor GM6001 could inhibit this effect. Using clinically relevant EGFR inhibitors, erlotinib and lapatinib, we found that inhibition of EGFR activation effectively inhibited leptin- and IGF-I-induced invasion and migration of breast cancer cells. Taken together, these data suggest a novel bidirectional crosstalk between leptin and IGF-I signaling that transactivates EGFR and promotes the metastatic properties as well as invasion and migration of breast cancer cells. Our findings indicate the possibility of using EGFR inhibitors erlotinib and lapatinib to counter the procancerous effects of leptin and IGF-I in breast cancers.