TRPV4 belongs to the ‘Transient Receptor Potential’ (TRP) superfamily. It has been identified to profoundly affect a variety of physiological processes, including nociception, heat sensation and inflammation. Unlike other TRP superfamily channels, its role in cancers are unknown until recently when we reported TRPV4 to be required for cancer cell softness that may promote breast cancer cell extravasation and metastasis. Here, we elucidated the molecular mechanisms mediated by TRPV4 in the metastatic breast cancer cells. TRPV4-mediated signaling was demonstrated to involve Ca²⁺-dependent activation of AKT and downregulation of E-cadherin expression, which was abolished upon TRPV4 silencing. Functionally, TRPV4-enhanced breast caner cell transendothelial migration requires AKT activity while a combination of transcriptional and post-translational regulation contributed to the TRPV4-mediated E-cadherin downregulation. Finally, mass spectrometry analysis revealed that TRPV4 is required for the expression of a network of secreted proteins involved in extracellular matrix remodeling. In conclusion, TRPV4 may regulate breast cancer metastasis by regulating cell softness through the Ca²⁺-dependent AKT-E-cadherin signaling axis and regulation of the expression of extracellular proteins.

Metastasis is a significant health issue. The standard mode of care is combination of chemotherapy and targeted therapeutics but the 5-year survival rate remains low. New/better drug targets that can improve outcomes of patients with metastatic disease are needed. Metastasis is a complex process, with each step conferred by a set of genetic aberrations. Mapping the molecular changes associated with metastasis improves our understanding of the etiology of this disease and contributes to the pipeline of targeted therapeutics. Here, phosphoproteomics of a xenograft-derived in vitro model comprising 4 isogenic cell lines with increasing metastatic potential implicated Transient Receptor Potential Vanilloid subtype 4 in breast cancer metastasis. TRPV4 mRNA levels in breast, gastric and ovarian cancers correlated with poor clinical outcomes, suggesting a wide role of TRPV4 in human epithelial cancers. TRPV4 was shown to be required for breast cancer cell invasion and transendothelial migration but not growth/proliferation. Knockdown of Trpv4 significantly reduced the number of metastatic nodules in mouse xenografts leaving the size unaffected. Overexpression of TRPV4 promoted breast cancer cell softness, blebbing, and actin reorganization. The findings provide new insights into the role of TRPV4 in cancer extravasation putatively by reducing cell rigidity through controlling the cytoskeleton at the cell cortex.

Cross-talk between the Hippo and Wnt pathways has been implicated recently in breast cancer development, but key intersections have yet to be fully defined. Here we report that WBP2, a transcription coactivator that binds the Hippo pathway transcription factor YAP/TAZ, contributes to Wnt signaling and breast cancer pathogenesis. Clinically, overexpression of WBP2 in breast cancer specimens correlated with malignant progression and poor patient survival. In breast cancer cells, nuclear entry and interaction of WBP2 with β-catenin was stimulated by Wnt3A, thereby activating TCF-mediated transcription and driving malignant invasive character. Mechanistic investigations showed WBP2 levels were controlled by the E3 ligase ITCH, which bound and target WBP2 for ubiquitin-dependent proteasomal degradation. Accordingly, ITCH silencing could elevate WBP2 levels. Wnt signaling upregulated WBP2 by disrupting ITCH–WBP2 interactions via EGFR-mediated tyrosine phosphorylation of WBP2 and TAZ/YAP competitive binding. Conversely, ITCH-mediated downregulation of WBP2 inhibited TCF/β-catenin transcription, in vitro transformation, and in vivo tumorigenesis. We identified somatic mutations in ITCH, which impaired its ability to degrade WBP2 and to block its function in cancer, even while retaining binding capacity to WBP2. Thus, the Wnt pathway appeared to engage WBP2 primarily by affecting its protein stability. Our findings show how WBP2/ITCH signaling functions to link the intricate Wnt and Hippo signaling networks in breast cancer. Cancer Res; 76(21); 6278–89. ©2016 AACR.

Secreted proteins are an attractive minefield for cancer drug targets. An iTRAQ-based tandem mass spectrometry approach was employed to relatively quantify proteins in the secretomes of four isogenic breast cancer cell lines with increasing metastatic potential. CXCL3 was found to be upregulated in aggressive cancer cells. SiRNA and antibody neutralization studies supported a role of CXCL3 in metastatic processes. Meta-analysis of the mRNA level of CXCL3 in 1881 breast tumors supported a role of CXCL3 in clinical breast cancer. Our results support a functional role of CXCL3 in breast cancer metastasis and as a viable target for cancer therapy.

Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process. Repressors and effectors form close interacting networks in pathways, including RNA processing, G protein signaling, protein ubiquitination, and chromatin modification. Combinatorial knockdown of five repressors (SMAD3, ZMYM2, SFRS11, SAE1, and ESET) synergistically resulted in ∼85% TRA-1-60-positive cells. Removal of the novel splicing factor SFRS11 during reprogramming is accompanied by rapid acquisition of pluripotency-specific spliced forms. Mechanistically, SFRS11 regulates exon skipping and mutually exclusive splicing of transcripts in genes involved in cell differentiation, mRNA splicing, and chromatin modification. Our study provides insights into the reprogramming process, which comprises comprehensive and multi-layered transcriptional, splicing, and epigenetic machineries.