HuR -dependent SOD2 protein synthesis is an early adaptation of ovarian cancer cells in response to anchorage independence

The presence of malignant ascites is a common feature of advanced stage ovarian cancer. During metastasis, as cells detach from the tumor and extravasate into the peritoneal fluid, ovarian cancer cells must adapt to survive the loss of anchorage support and evade anoikis. An important pro-survival adaptation in this context is the ability of tumor cells to increase their antioxidant capacity and restore cellular redox balance. We previously showed that the mitochondrial superoxide dismutase SOD2 is necessary for ovarian cancer cell anoikis resistance, anchorage-independent survival and spheroid formation, and intraperitoneal spread in vivo. We now demonstrate that the upregulation of SOD2 protein expression is an early event initiated in response to anchorage independence and occurs at the post-transcriptional level. SOD2 protein synthesis is rapidly induced in the cytosol within 2 hours of matrix detachment. Polyribosome profiling demonstrates an increase in the number of ribosomes bound to SOD2 mRNA, indicating an increase in SOD2 translation in response to anchorage-independence. Mechanistically, we find that anchorage-independence induces cytosolic accumulation of the RNA binding protein HuR/ELAVL1, leads to HuR binding to SOD2 mRNA, and that the presence of HuR is necessary for the increase in SOD2 mRNA association with the heavy polyribosome fraction and SOD2 protein synthesis. Cellular detachment activates the stress-response protein kinase p38 MAPK, which is necessary for HuR-SOD2 mRNA binding and the rapid increase in SOD2 protein expression. Moreover, HuR is necessary for optimal cell survival during early stages of anchorage independence. These findings uncover a novel post-transcriptional stress response mechanism by which tumor cells are able to rapidly increase their mitochondrial antioxidant capacity to adapt to stress associated with anchorage-independence.

FOXA1 is a determinant of drug resistance in breast cancer cells

Purpose Breast cancer is one of the most commonly diagnosed cancers in women. Five subtypes of breast cancer differ in their genetic expression profiles and carry different prognostic values, with no treatments available for some types, such as triple-negative, due to the absence of genetic signatures that could otherwise be targeted by molecular therapies. Although endocrine treatments are largely successful for estrogen receptor (ER)-positive cancers, a significant proportion of patients with metastatic tumors fail to respond and acquire resistance to therapy. FOXA1 overexpression mediates endocrine therapy resistance in ER-positive breast cancer, although the regulation of chemotherapy response by FOXA1 has not been addressed previously. FOXA1, together with EP300 and RUNX1, regulates the expression of E-cadherin, and is expressed in luminal, but absent in triple-negative and basal-like breast cancers. We have previously determined that EP300 regulates drug resistance and tumor initiation capabilities in breast cancer cells. Methods Here we describe the generation of breast cancer cell models in which FOXA1 expression has been modulated either by expression of hairpins targeting FOXA1 mRNA or overexpression plasmids. Results Upon FOXA1 knockdown in luminal MCF-7 and T47D cells, we found an increase in doxorubicin and paclitaxel sensitivity as well as a decrease in anchorage independence. Conversely, upregulation of FOXA1 in basal-like MDA-MB-231 cells led to an increase in drug resistance and anchorage independence. Conclusion Together, these data suggest that FOXA1 plays a role in making tumors more aggressive.

Publisher Correction: B4GALNT1 induces angiogenesis, anchorage independence growth and motility, and promotes tumorigenesis in melanoma by induction of ganglioside GM2/GD2

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B4GALNT1 induces angiogenesis, anchorage independence growth and motility, and promotes tumorigenesis in melanoma by induction of ganglioside GM2/GD2

β-1,4-N-Acetyl-Galactosaminyltransferase 1 (B4GALNT1) encodes the key enzyme B4GALNT1 to generate gangliosides GM2/GD2. GM2/GD2 gangliosides are surface glycolipids mainly found on brain neurons as well as peripheral nerves and skin melanocytes and are reported to exacerbate the malignant potential of melanomas. In order to elucidate the mechanism, we performed functional analyses of B4GALNT1-overexpressing cells. We analyzed ganglioside pattern on four melanoma and two neuroblastoma cell lines by high performance liquid chromatography (HPLC). We overexpressed B4GALNT1 in GM2/GD2-negative human melanoma cell line (SH4) and confirmed production of GM2/GD2 by HPLC. They showed higher anchorage independence growth (AIG) in colony formation assay, and exhibited augmented motility. In vitro, cell proliferation was not affected by GM2/GD2 expression. In vivo, GM2/GD2-positive SH4 clones showed significantly higher tumorigenesis in NOD/Scid/IL2Rγ-null mice, and immunostaining of mouse CD31 revealed that GM2/GD2 induced remarkable angiogenesis. No differences were seen in melanoma stem cell and Epithelial-Mesenchymal Transition markers between GM2/GD2-positive and -negative SH4 cells. We therefore concluded that B4GALNT1, and consequently GM2/GD2, enhanced tumorigenesis via induction of angiogenesis, AIG, and cell motility. RNA-Seq suggested periostin as a potential key factor for angiogenesis and AIG. These findings may lead to development of novel therapy for refractory melanoma.

Proteomic and Transcriptomic Profiling Identifies Mediators of Anchorage-Independent Growth and Roles of Inhibitor of Differentiation Proteins in Invasive Lobular Breast Cancer

BACKGROUNDInvasive lobular carcinoma (ILC) is a histological subtype of breast cancer with distinct molecular and clinical features from the more common subtype invasive lobular carcinoma (IDC). We have previously shown that human ILC cells lines have a remarkably unique ability to grow in ultra-low attachment (ULA) suspension cultures as compared to IDC cells, the mediators of which remain unknown.METHODSUsing flow cytometry and immunoblotting in human ILC and IDC cell lines, we measured levels of apoptosis and cell proliferation in attached (2D) and suspension (ULA) cultures. siRNA-mediated knockdown and pharmacological inhibitors were utilized to assess the effects of known regulators of anchorage-independence. Reverse Phase Protein Arrays and RNA-Sequencing were performed to identify novel proteomic and transcriptomic mediators of ULA growth in ILC cells.RESULTSWe show that human ILC cell lines exhibit enhanced anoikis resistance and cell proliferation in ULA cultures as compared to IDC cells. Transient restoration of E-cadherin did not impact the 2D or ULA growth of human ILC cell lines, while transient E-cadherin knockdown in IDC cells partially rescued their growth defect in ULA culture. Inhibition of the Rho/ROCK, p120-catenin or YAP/Hippo pathways previously implicated in anoikis resistance did not have a major effect on the ULA growth of ILC cells. Proteomic comparison of ILC and IDC cell lines identified unique induction of PI3K/Akt and p90-RSK pathways in ULA culture in ILC cells. 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 anchorage-independent growth. We find that ID1 and ID3 expression is higher in human ILC tumors as compared to IDC and correlated with a worse disease-specific survival uniquely in the ILC cohort.CONCLUSIONOur comprehensive study of 2D and ULA growth in human ILC cell lines revealed anoikis resistance, cell proliferation and novel mediators of anchorage-independence and provides possible mechanistic insights and clinical implications for metastatic dissemination of ILC. High expression in human ILC tumors and association with clinical outcome implicate ID1 and ID3 as novel drivers and therapeutic targets for lobular breast cancer.