VEGFA links self-renewal and metastasis by inducing Sox2 to repress miR-452, driving Slug.

Cancer stem cells (CSC) appear to have increased metastatic potential, but mechanisms underlying this are poorly defined. Here we show that VEGFA induction of Sox2 promotes EMT and tumor metastasis. In breast lines and primary cancer culture, VEGFA rapidly upregulates SOX2 expression, leading to SNAI2 induction, EMT, increased invasion and metastasis. We show Sox2 downregulates miR-452, which acts as a novel metastasis suppressor to directly target the SNAI2 3'-untranslated region (3'-UTR). VEGFA stimulates Sox2- and Slug-dependent cell invasion. VEGFA increases lung metastasis in vivo, and this is abrogated by miR-452 overexpression. Furthermore, SNAI2 transduction rescues metastasis suppression by miR-452. Thus, in addition to its angiogenic action, VEGFA upregulates Sox2 to drive stem cell expansion, together with miR-452 loss and Slug upregulation, providing a novel mechanism whereby cancer stem cells acquire metastatic potential. Prior work showed EMT transcription factor overexpression upregulates CSC. Present work indicates that stemness and metastasis are a two-way street: Sox2, a major mediator of CSC self-renewal, also governs the metastatic process.

Validation of suitable normalizers for miR expression patterns analysis covering tumour heterogeneity.

Oncogenic microRNAs (miRs) have emerged as diagnostic biomarkers and novel molecular targets for anti-cancer drug therapies. Real-time quantitative PCR (qPCR) is one of the most powerful techniques for analyzing miRs; however, the use of unsuitable normalizers might bias the results. Tumour heterogeneity makes even more difficult the selection of an adequate endogenous normalizer control. Here, we have evaluated five potential referenced small RNAs (U6, rRNA5s, SNORD44, SNORD24 and hsa-miR-24c-3p) using RedFinder algorisms to perform a stability expression analysis in i) normal colon cells, ii) colon and breast cancer cell lines and iii) cancer stem-like cell subpopulations. We identified SNORD44 as a suitable housekeeping gene for qPCR analysis comparing normal and cancer cells. However, this small nucleolar RNA was not a useful normalizer for cancer stem-like cell subpopulations versus subpopulations without stemness properties. In addition, we show for the first time that hsa-miR-24c-3p is the most stable normalizer for comparing these two subpopulations. Also, we have identified by bioinformatic and qPCR analysis, different miR expression patterns in colon cancer versus non tumour cells using the previously selected suitable normalizers. Our results emphasize the importance of select suitable normalizers to ensure the robustness and reliability of qPCR data for analyzing miR expression.

Vitamin D and androgen receptor-targeted therapy for triple-negative breast cancer.

Anti-estrogen and anti-HER2 treatments have been among the first and most successful examples of targeted therapy for breast cancer (BC). However, the treatment of triple-negative BC (TNBC) that lack estrogen receptor expression or HER2 amplification remains a major challenge. We previously discovered that approximately two-thirds of TNBCs express vitamin D receptor (VDR) and/or androgen receptor (AR) and hypothesized that TNBCs co-expressing AR and VDR (HR2-av TNBC) could be treated by targeting both of these hormone receptors. To evaluate the feasibility of VDR/AR-targeted therapy in TNBC, we characterized 15 different BC lines and identified 2 HR2-av TNBC lines and examined the changes in their phenotype, viability, and proliferation after VDR and AR-targeted treatment. Treatment of BC cell lines with VDR or AR agonists inhibited cell viability in a receptor-dependent manner, and their combination appeared to inhibit cell viability additively. Moreover, cell viability was further decreased when AR/VDR agonist hormones were combined with chemotherapeutic drugs. The mechanisms of inhibition by AR/VDR agonist hormones included cell cycle arrest and apoptosis in TNBC cell lines. In addition, AR/VDR agonist hormones induced differentiation and inhibited cancer stem cells (CSCs) measured by reduction in tumorsphere formation efficiency, high aldehyde dehydrogenase activity, and CSC markers. Surprisingly, we found that AR antagonists inhibited proliferation of most BC cell lines in an AR-independent manner, raising questions regarding their mechanism of action. In summary, AR/VDR-targeted agonist hormone therapy can inhibit HR2-av TNBC through multiple mechanisms in a receptor-dependent manner and can be combined with chemotherapy.

VEGF drives cancer-initiating stem cells through VEGFR-2/Stat3 signaling to upregulate Myc and Sox2.

Vascular endothelial growth factor-A (VEGF), a potent angiogenic factor, is also implicated in self-renewal in several normal tissue types. VEGF has been shown to drive malignant stem cells but mechanisms thereof and tumor types affected are not fully characterized. Here, we show VEGF promotes breast and lung cancer stem cell (CSC) self-renewal via VEGF receptor-2 (VEGFR-2)/STAT3-mediated upregulation of Myc and Sox2. VEGF increased tumor spheres and aldehyde dehydrogenase activity, both proxies for stem cell function in vitro, in triple-negative breast cancer (TNBC) lines and dissociated primary cancers, and in lung cancer lines. VEGF exposure before injection increased breast cancer-initiating cell abundance in vivo yielding increased orthotopic tumors, and increased metastasis from orthotopic primaries and following tail vein injection without further VEGF treatment. VEGF rapidly stimulated VEGFR-2/JAK2/STAT3 binding and activated STAT3 to bind MYC and SOX2 promoters and induce their expression. VEGFR-2 knockdown or inhibition abrogated VEGF-mediated STAT3 activation, MYC and SOX2 induction and sphere formation. Notably, knockdown of either STAT3, MYC or SOX2 impaired VEGF-upregulation of pSTAT3, MYC and SOX2 expression and sphere formation. Each transcription factor, once upregulated, appears to promote sustained activation of the others, creating a feed-forward loop to drive self-renewal. Thus, in addition to angiogenic effects, VEGF promotes tumor-initiating cell self-renewal through VEGFR-2/STAT3 signaling. Analysis of primary breast and lung cancers (>1300 each) showed high VEGF expression, was prognostic of poor outcome and strongly associated with STAT3 and MYC expression, supporting the link between VEGF and CSC self-renewal. High-VEGF tumors may be most likely to escape anti-angiogenics by upregulating VEGF, driving CSC self-renewal to re-populate post-treatment. Our work highlights the need to better define VEGF-driven cancer subsets and supports further investigation of combined therapeutic blockade of VEGF or VEGFR-2 and JAK2/STAT3.