BHLHE40 confers a pro-survival and pro-metastatic phenotype to breast cancer cells by modulating HBEGF secretion

Abstract Background Metastasis is responsible for a significant number of breast cancer-related deaths. Hypoxia, a primary driving force of cancer metastasis, induces the expression of BHLHE40, a transcription regulator. This study aimed to elucidate the function of BHLHE40 in the metastatic process of breast cancer cells. Methods To define the role of BHLHE40 in breast cancer, BHLHE40 expression was knocked down by a lentiviral construct expressing a short hairpin RNA against BHLHE40 or knocked out by the CRISPR/Cas9 editing system. Orthotopic xenograft and experimental metastasis (tail vein injection) mouse models were used to analyze the role of BHLHE40 in lung metastasis of breast cancer. Global gene expression analysis and public database mining were performed to identify signaling pathways regulated by BHLHE40 in breast cancer. The action mechanism of BHLHE40 was examined by chromatin immunoprecipitation (ChIP), co-immunoprecipitation (CoIP), exosome analysis, and cell-based assays for metastatic potential. Results BHLHE40 knockdown significantly reduced primary tumor growth and lung metastasis in orthotopic xenograft and experimental metastasis models of breast cancer. Gene expression analysis implicated a role of BHLHE40 in transcriptional activation of heparin-binding epidermal growth factor (HBEGF). ChIP and CoIP assays revealed that BHLHE40 induces HBEGF transcription by blocking DNA binding of histone deacetylases (HDAC)1 and HDAC2. Cell-based assays showed that HBEGF is secreted through exosomes and acts to promote cell survival and migration. Public databases provided evidence linking high expression of BHLHE40 and HBEGF to poor prognosis of triple-negative breast cancer. Conclusion This study reveals a novel role of BHLHE40 in promoting tumor cell survival and migration by regulating HBEGF secretion

The natural compound Jatrophone interferes with Wnt/β-catenin signaling and inhibits proliferation and EMT in human triple-negative breast cancer

<div><p>Metastatic breast cancer is the leading cause of worldwide cancer-related deaths among women. Triple negative breast cancers (TNBC) are highly metastatic and are devoid of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) amplification. TNBCs are unresponsive to Herceptin and/or anti-estrogen therapies and too often become highly chemoresistant when exposed to standard chemotherapy. TNBCs frequently metastasize to the lung and brain. We have previously shown that TNBCs are active for oncogenic <i>Wnt10b/β-catenin</i> signaling and that WNT10B ligand and its downstream target HMGA2 are predictive of poorer outcomes and are strongly associated with chemoresistant TNBC metastatic disease. In search of new chemicals to target the oncogenic WNT10B/β-CATENIN/HMGA2 signaling axis, the anti-proliferative activity of the diterpene Jatrophone (JA), derived from the plant <i>Jatropha isabelli</i>, was tested on TNBC cells. JA interfered with the WNT TOPFLASH reporter at the level between receptor complex and β-catenin activation. JA efficacy was determined in various subtypes of TNBC conventional cell lines or in TNBC cell lines derived from TNBC PDX tumors. The differential IC₅₀ (DCI₅₀) responsiveness was compared among the TNBC models based on etiological-subtype and their cellular chemoresistance status. Elevated <i>WNT10B</i> expression also coincided with increased resistance to JA exposure in several metastatic cell lines. JA interfered with cell cycle progression, and induced loss of expression of the canonical Wnt-direct targets genes AXIN2, HMGA2, MYC, PCNA and CCND1. Mechanistically, JA reduced steady-state, non-phosphorylated (activated) β-catenin protein levels, but not total β-catenin levels. JA also caused the loss of expression of key EMT markers and significantly impaired wound healing in scratch assays, suggesting a direct role for JA inhibiting migration of TNBC cells. These results indicate that Jatrophone could be a powerful new chemotherapeutic agent against highly chemoresistant triple negative breast cancers by targeting the oncogenic <i>Wnt10b/β-catenin</i> signaling pathway.</p></div

JA inhibits EMT-markers and prevents cell migration in wound healing assays.

<p><b>(A)</b> qPCR for <i>SLUG</i>, <i>FIBRONETIN</i>, <i>VIMENTIN</i> and <i>ZEB1</i> in ICG-001 and JA treated MDA-MB-231 cells. Results are expressed as mean ± SE, <i>n</i> = 3 *p<0.05. <b>(B)</b> The number of cells migrated into the scratched area was photographed (340) and calculated as a percentage of migration for 16 hours post treatment. Quantification of triplicates using <i>t-test</i>: ***-p < 0.001 vs. control.</p

Determination of differential IC₅₀ (DIC₅₀) effects of JA on various TNBC cell lines and TNBC PDX-derived cell lines.

<p>WST-1 proliferation assays for 48 hours utilizing JA at various dosages ranging from 100 nM-30 μM: <b>(A)</b> MSL-subtype TNBC cell lines MDA-MB-231 (EA) vs. MDA-MB-157 (AA). <b>(B)</b> BL1-subtype TNBC cell lines HCC-38 (EA) vs. MDA-MB-468 (AA). <b>(C)</b> TNBC PDX-derived “naïve” cell line HCl-2 vs. “chemoresistant” HCl-10. The IC₅₀ of JA indicated for each cell lines determined by n = 3, in triplicate, using the two-tailed <i>t-test p</i>-values were calculated: *p < 0.05, **-p < 0.01 *** < 0.001 relative to vehicle control cells (DMSO).</p

Compounds JA arrests cells at S-phase of cell cycle and induced apoptosis in MDA-MB-231 Cells.

<p><b>A)</b> Annexin V-FITC staining was used to detect apoptosis by flow analysis in control (DMSO), staurosporine (1μM), ICG-001 (10 μM) and JA (2.5 μM) treated cells for 48 hours. Cells were counter stain with propidium iodide (PI, 1 μg/ml). We show one representative FACS plot <b>B)</b> Quantification of alive, early and late apoptotic cells in cells from panel A. <b>C)</b> qPCR for Survivin (<i>BIRC5</i>) in control cells, ICG-001 and JA treated cells. <b>(D)</b> One representative FACS plot using PI/DNA content analyzed by flow cytometry <b>(E)</b> Quantification of cell cycle phases for in G₀/G₁, S and G₂/M in both ICG-001 and JA treated cells. <b>(F)</b> qPCR for cell cycle markers <i>CDK4</i>, <i>CCND1</i>, <i>CCNE1</i>, <i>CCNA1 and CCNB1</i> from the same cells as in panel E. Statistics on three biological independent experiments with duplicates for each of the above. <i>T-test</i> was used to determine <i>p</i>-values: ***-p < 0.001, **-p < 0.01 and *p < 0.05 vs. control.</p

JA inhibits expression on Wnt/β-catenin direct-target genes and degrades non-phosphorylated activated β-catenin protein levels.

<p><b>(A)</b> qPCR for <i>BIRC5</i>, <i>AXIN2</i>, <i>HMGA2</i>, <i>CNND1</i>, <i>MYC</i> and <i>PCNA</i> in ICG-001 and JA treated MDA-MB-231 cells. <b>(B)</b> Immunoblot analysis for AXIN2, HMGA2, CYCLIND1, PCNA and MYC. ACTIN serves as the loading control. <b>(C)</b> Immunoblot analysis for non-phosphorylated activated β-CATENIN (ABC), total pan-β-CATENIN and GS3Kβ^Ser9 from the same cells and extracts as in panel A and B. Quantification of triplicates using <i>t-test</i> p values are ***-p < 0.001, **-p < 0.01 and *p < 0.05 vs. control.</p