Canonical Wnt signaling is antagonized by noncanonical Wnt5a in hepatocellular carcinoma cells

<p>Abstract</p> <p>Background</p> <p>β-catenin mutations that constitutively activate the canonical Wnt signaling have been observed in a subset of hepatocellular carcinomas (HCCs). These mutations are associated with chromosomal stability, low histological grade, low tumor invasion and better patient survival. We hypothesized that canonical Wnt signaling is selectively activated in well-differentiated, but repressed in poorly differentiated HCCs. To this aim, we characterized differentiation status of HCC cell lines and compared their expression status of Wnt pathway genes, and explored their activity of canonical Wnt signaling.</p> <p>Results</p> <p>We classified human HCC cell lines into "well-differentiated" and "poorly differentiated" subtypes, based on the expression of hepatocyte lineage, epithelial and mesenchymal markers. Poorly differentiated cell lines lost epithelial and hepatocyte lineage markers, and overexpressed mesenchymal markers. Also, they were highly motile and invasive. We compared the expression of 45 Wnt pathway genes between two subtypes. TCF1 and TCF4 factors, and LRP5 and LRP6 co-receptors were ubiquitously expressed. Likewise, six Frizzled receptors, and canonical Wnt3 ligand were expressed in both subtypes. In contrast, canonical ligand Wnt8b and noncanonical ligands Wnt4, Wnt5a, Wnt5b and Wnt7b were expressed selectively in well- and poorly differentiated cell lines, respectively. Canonical Wnt signaling activity, as tested by a TCF reporter assay was detected in 80% of well-differentiated, contrary to 14% of poorly differentiated cell lines. TCF activity generated by ectopic mutant β-catenin was weak in poorly differentiated SNU449 cell line, suggesting a repressive mechanism. We tested Wnt5a as a candidate antagonist. It strongly inhibited canonical Wnt signaling that is activated by mutant β-catenin in HCC cell lines.</p> <p>Conclusion</p> <p>Differential expression of Wnt ligands in HCC cells is associated with selective activation of canonical Wnt signaling in well-differentiated, and its repression in poorly differentiated cell lines. One potential mechanism of repression involved Wnt5a, acting as an antagonist of canonical Wnt signaling. Our observations support the hypothesis that Wnt pathway is selectively activated or repressed depending on differentiation status of HCC cells. We propose that canonical and noncanonical Wnt pathways have complementary roles in HCC, where the canonical signaling contributes to tumor initiation, and noncanonical signaling to tumor progression.</p

Bromodomain protein BRD4 is a transcriptional repressor of autophagy and lysosomal function

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation

Proteomic and transcriptomic profiling identifies mediators of anchorage-independent growth and roles of inhibitor of differentiation proteins in invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer with distinct molecular and clinical features from the more common subtype invasive ductal carcinoma (IDC). ILC cells exhibit anchorage-independent growth in ultra-low attachment (ULA) suspension cultures, which is largely attributed to the loss of E-cadherin. In addition to anoikis resistance, herein we show that human ILC cell lines exhibit enhanced cell proliferation in ULA cultures as compared to IDC cells. Proteomic comparison of ILC and IDC cell lines identified induction of PI3K/Akt and p90-RSK pathways specifically in ULA culture in ILC cells. Further transcriptional profiling uncovered unique upregulation of the inhibitors of differentiation family transcription factors ID1 and ID3 in ILC ULA culture, the knockdown of which diminished the anchorage-independent growth of ILC cell lines through cell cycle arrest. We find that ID1 and ID3 expression is higher in human ILC tumors as compared to IDC, correlated with worse prognosis uniquely in patients with ILC and associated with upregulation of angiogenesis and matrisome-related genes. Altogether, our comprehensive study of anchorage independence in human ILC cell lines provides mechanistic insights and clinical implications for metastatic dissemination of ILC and implicates ID1 and ID3 as novel drivers and therapeutic targets for lobular breast cancer

Differential expression of full-length and NH₂ terminally truncated FAM134B isoforms in normal physiology and cancer.

Selective autophagy of the endoplasmic reticulum (ER), namely ER-phagy, is mediated by ER-localized receptors, which are recognized and sequestered by GABARAP/LC3B-decorated phagophores and transferred to lysosomes for degradation. Being one such receptor, FAM134B plays critical roles in cellular processes such as protein quality control and neuronal survival. FAM134B has also been associated with different cancers, although its exact role remains elusive. We report here that the FAM134B gene encodes not one but at least two different protein isoforms: the full-length and the NH2 terminally truncated forms. Their relative expression shows extreme variation, both within normal tissues and among cancer types. Expression of full-length FAM134B is restricted to the brain, testis, spleen, and prostate. In contrast, NH2 terminally truncated FAM134B is dominant in the heart, skeletal muscle, kidney, pancreas, and liver. We compared wild-type and knockout mice to study the role of the Fam134b gene in starvation. NH2 terminally truncated FAM134B-2 was induced in the liver, skeletal muscle, and heart but not in the pancreas and stomach following starvation. Upon starvation, Fam134b(-/-) mice differed from wild-type mice by less weight loss and less hyperaminoacidemic and hypocalcemic response but increased levels of serum albumin, total serum proteins, and a-amylase. Interestingly, either NH2 terminally truncated FAM134B or both isoforms were downregulated in liver, lung, and colon cancers. In contrast, upregulation was observed in stomach and chromophobe kidney cancers

Mendelian transmission of Rosa26-targted CAGs-rtTA3 transgenes.

<p>*Numbers represent: observed (expected) from heterzygote x wild-type crosses.</p

CAGs-rtTA3 and CAGs-RIK show strong expression in adult tissues.

<p>Whole mount epifluorescence images of small intestine, skin, pancreas kidney and liver from <i>R26-rtTA</i>, <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> transgenic animals (all containing <i>TG-Ren.713</i>). <i>R26-rtTA</i> shows strong expression in intestine and skin but weak or patchy expression in most other solid organs. <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> show almost identical expression patterns in adult mice. <i>CAGs-RIK</i> mice show strong and consistent expression of mKate2.</p

<i>CAGs-LSL-RIK</i> enables tissue-restricted expression of <i>TRE</i>-transgenes in transgenic models of disease.

<p><b>A</b>. Whole mount epifluorescence (top panel) and immunofluorescence images from a quadruple transgenic (<i>CAGs-LSL-RIK;TG-Ren.713;LSL-Kras^G12D;Pdx1-Cre</i>) animal, showing induction of GFP and mKate2 in both normal acinar tissue and pre-neoplastic, Kras^G12D-induced PanIN lesions (top arrow). As observed in AdenoCre treated lungs, some PanIN lesions did not show GFP or mKate2 staining suggesting incomplete LSL excision in a small proportion of cells. <b>B</b>. Immunofluorescent stains for GFP and mKate2 in mammary tissue of <i>CAGs-LSL-RIK;TG-Ren.713;MMTV-Neu;WAP-Cre</i> transgenic mice treated with dox.</p

GFP induction and mKate2 expression is uniform in most organs of <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> mice.

<p>Immunofluorescence stains for GFP and mKate2 in the small intestine and pancreas of ‘no rtTA’, <i>R26-rtTA</i>, <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> mice following 1 week of doxycycline treatment. All rtTA strains show strong GFP induction in small intestine (<b>A</b>), but only <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> show robust and uniform GFP expression (and mKate2 for <i>RIK</i>) in the pancreatic acinar tissue (<b>B</b>).</p