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 s

ABSTRACT 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. Reexpression 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

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

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

Snail Promotes Epithelial Mesenchymal Transition in Breast Cancer Cells in Part via Activation of Nuclear ERK2

<div><p>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 <u>were</u> 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 <u>suggest</u> 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.</p></div

Snail promotes EMT via ERK2.

<p>Cells with low Snail maintain an epithelial morphology. During cancer progression, the expression of Snail <u>increases</u>, which promotes the nuclear localization of p-ERK2 isoform. This leads to EMT characterized by decreased E-cadherin, increased vimentin, decreased cell adhesion and increased cell migration. By a positive feedback loop, p-ERK can increase Snail expression. This leads to tumors with high levels of Snail and a mesenchymal morphology.</p

p-ERK expression is increased in patient tumor tissues.

<p>(A) 10 µg of normal/tumor-matched infiltrating ductal carcinoma (IDC) grades 1–3 and lymph node metastatic patient lysates were separated using SDS-PAGE electrophoresis, then immunoblotted onto nitrocellulose. Expression of p-ERK and ERK was determined using Western blot analysis. β-actin was used as Western blot loading control. (B) Human breast cancer tissue microarray was double-labeled with p-ERK (green) and Snail (red) antibodies using immunofluorescence analysis. DAPI was used to identify the nuclei. Images were captured using Zeiss Axiovision Rel4.8 <u>at 20× (left panel</u>) and Apotome software at and 40× oil magnification <u>(right panel)</u>. Results are representative of at least three independent experiments.</p

Inhibition of p-ERK with UO126 decreases Snail and reverts EMT independent of proteasomal degradation.

<p>(A) Expression of p-ERK, ERK and Snail was analyzed by Western blot analysis in MCF-7 Neo and MCF-7 Snail cells treated with DMSO control (Ctrl) or UO126 for 30 min, 2 h, 6 h, and 24 h. (B) MCF-7 Neo and MC-7 Snail cells <u>were treated with either DMSO (control) or UO126 and stained with DAPI</u>. Cell morphology and integrity were analyzed by merging DAPI immunofluorescence imaging with brightfield microscopy. (C) Expression of E-cadherin and vimentin in cells treated with DMSO Ctrl or UO126 was analyzed by Western blot analysis. (D) MCF-7 Snail were treated for 6 h and 24 h with DMSO Ctrl, UO126 and UO126+MG132. Cell lysates were analyzed by Western blot analysis. (E) Migration and (F) adhesion assays was performed on MCF-7 Neo and MCF-7 Snail <u>cells</u> treated with DMSO Ctrl or UO126. β-actin was utilized as a loading control for Western blot analysis; DAPI was used to identify the nuclei in immunofluorescence analyses. <u>Maginification 40X</u>. <u>Statistical Analysis was done using ANOVA and Tukey's Multiple Comparison as Post Hoc (**p≤0.01, ***p≤0.001). Values were expressed as mean ± S.E.M (N = 3)</u>. Results are representative of at least three independent experiments.</p