PEAK1 Acts as a Molecular Switch to Regulate Context-Dependent TGFβ Responses in Breast Cancer

<div><p>Transforming Growth Factor β (TGFβ) has dual functions as both a tumor suppressor and a promoter of cancer progression within the tumor microenvironment, but the molecular mechanisms by which TGFβ signaling switches between these outcomes and the contexts in which this switch occurs remain to be fully elucidated. We previously identified PEAK1 as a new non-receptor tyrosine kinase that associates with the cytoskeleton, and facilitates signaling of HER2/Src complexes. We also showed PEAK1 functions downstream of KRas to promote tumor growth, metastasis and therapy resistance using preclinical <i>in vivo</i> models of human tumor progression. In the current study, we analyzed PEAK1 expression in human breast cancer samples and found PEAK1 levels correlate with mesenchymal gene expression, poor cellular differentiation and disease relapse. At the cellular level, we also observed that PEAK1 expression was highest in mesenchymal breast cancer cells, correlated with migration potential and increased in response to TGFβ-induced epithelial-mesenchymal transition (EMT). Thus, we sought to evaluate the role of PEAK1 in the switching of TGFβ from a tumor suppressing to tumor promoting factor. Notably, we discovered that high PEAK1 expression causes TGFβ to lose its anti-proliferative effects, and potentiates TGFβ-induced proliferation, EMT, cell migration and tumor metastasis in a fibronectin-dependent fashion. In the presence of fibronectin, PEAK1 caused a switching of TGFβ signaling from its canonical Smad2/3 pathway to non-canonical Src and MAPK signaling. This report is the first to provide evidence that PEAK1 mediates signaling cross talk between TGFβ receptors and integrin/Src/MAPK pathways and that PEAK1 is an important molecular regulator of TGFβ-induced tumor progression and metastasis in breast cancer. Finally, PEAK1 overexpression/upregulation cooperates with TGFβ to reduce breast cancer sensitivity to Src kinase inhibition. These findings provide a rational basis to develop therapeutic agents to target PEAK1 expression/function or upstream/downstream pathways to abrogate breast cancer progression.</p></div

PEAK1 is necessary and sufficient for increased breast cancer cell migration in response to TGFβ/fibronectin treatment.

<p>(A-D) MCF7-Vector and–PEAK1 or CA1h-shCntrl and–shPEAK1 cells were chronically treated with TGFβ while being cultured on plastic. (A and C) Single cell migration assays were performed on these cells after plating on the indicated substrates with 3 images per condition being collected every 10 minutes for 24 hours. Cells were tracked using Fiji software to determine Displacement (μm) (left y-axis) and Velocity (μm/min) (right y-axis). Two-way ANOVA statistical analysis was performed using Prism. (B and D) 10 representative cell tracks for each of the indicated cell populations are shown when cells were migrating on fibronectin. *** indicate p-values < 0.001.</p

PEAK1 is required for TGFβ/fibronectin-mediated metastasis <i>in vivo</i>.

<p>(A and B) CA1h-shCntrl and–shPEAK1 cells were plated on fibronectin then treated with TGFβ for 72 hours, after which a CAM assay was preformed. (A) After harvesting the primary tumor, it was weighed. (B) qPCR for chicken GAPDH and human <i>alu</i> was performed on genomic DNA extracted from the liver and lung tissue. A t-test was performed to determine statistical significance. *, **, *** indicate p-values < 0.05, 0.01 and 0.001, respectively.</p

PEAK1 cooperates with fibronectin to block the cytostatic effects of TGFβ.

<p>MCF7-Vector and–PEAK1 or CA1h-shCntrl and–shPEAK1 cells were plated on fibronectin then treated with TGFβ. (A and C) After 72 hour incubation, an AQueous One assay was performed. The relative cell number was plotted and two-way ANOVA analysis was performed to determine statistical significance. (B and D) The cell cycle profiles were analyzed and the percent of cells in each stage are shown. *, **, *** indicate p-values < 0.05, 0.01 and 0.001, respectively.</p

PEAK1 potentiates fibronectin/TGFβ-induced EMT-like responses.

<p>(A-C) MCF7-Vector and-PEAK1 cells were plated on 3ug/mL Fibronectin (F), Collagen (C) or Laminin (L), or plated on Plastic (P) and treated chronically with TGFβ. (A) Western blot analysis for E-cadherin, N-cadherin, and α-tubulin was performed after two weeks in culture. (B) Western blot band quantification from (A) using densitometry analysis. (C) Micrographs of cells after two weeks in culture. Yellow arrowhead points to a typical cell that is not packed tightly into epithelial colonies and is mesenchymal. (D & E) qPCR for E-Cadherin and Vimentin expression (D) and micrographs (E) from CA1h-shCntrl and-shPEAK1 cells plated on fibronectin and treated with TGFβ or vehicle control for 48 hours. (Scale bar: 90μm). Yellow arrowhead points to a typical cell that is not packed tightly into epithelial colonies and is mesenchymal.</p

PEAK1 and TGFβ cooperate to promote resistance to Src kinase inhibition.

<p>(A) Indicated populations of MCF7-Vector and–PEAK1 cells were plated on fibronectin and treated with increasing doses of either AZM (left) or SB-431542 (right). After 72 hours an AQueous One assay was performed to assess cell viability. Relative cell number versus drug concentration (M) is plotted. (B) AZM IC₅₀ values for are reported for the indicated cell population.</p

PEAK1 expression in breast cancer correlates with indicators of poor patient prognosis, mesenchymal gene expression, cell migration and is upregulated during TGFβ-induced EMT.

<p>(A) PEAK1 mRNA fold change was analyzed from several previous reports of patient data in relation to characteristics that correlate with poor patient prognosis–i.e., metastatic lesions, disease relapse, advanced N stage, high grade, HER2 positive status, and stromal-derived poor prognostic (SDPP) status. (B) IHC from the Human Cancer Atlas of four different patients–two with elevated PEAK1 levels and two with low PEAK1 levels–for SNAI1, FN1, OCLN and ESR1 expression. (C) Western blot analysis on lysates from MCF10A, MCF10AT1K, MCF10CA1h, and MCF10CA1a cells for PEAK1 and E-cadherin expression. (D) Single cell migration assay on 3μg/mL of Fibronectin (F), Collagen (C), or Laminin (L) of CA1h and CA1a cells (velocity is plotted on the left axis and displacement is plotted on the right). (E) MCF10A, CA1h, MDA-MB-231, and MCF7 cells were plated on plastic and treated for 72 hours with TGFβ. RNA was collected and qPCR for E-cadherin (top) and PEAK1 (bottom) expression was performed. ** or *** indicate p-values < 0.01 or 0.001, respectively.</p