Genome-wide association studies have identified thousands of SNPs associated with predisposition to various diseases, including prostate cancer. However, the mechanistic roles of these SNPs remain poorly defined, particularly for noncoding polymorphisms. Here we find that the prostate cancer risk-associated SNP rs339331 at 6q22 lies within a functional HOXB13-binding site. The risk-associated T allele at rs339331 increases binding of HOXB13 to a transcriptional enhancer, conferring allele-specific upregulation of the rs339331-associated gene RFX6. Suppression of RFX6 diminishes prostate cancer cell proliferation, migration and invasion. Clinical data indicate that RFX6 upregulation in human prostate cancers correlates with tumor progression, metastasis and risk of biochemical relapse. Finally, we observe a significant association between the risk-associated T allele at rs339331 and increased RFX6 mRNA levels in human prostate tumors. Together, our results suggest that rs339331 affects prostate cancer risk by altering RFX6 expression through a functional interaction with the prostate cancer susceptibility gene HOXB13.
The cell surface receptor CED-1 mediates apoptotic cell recognition by phagocytic cells, enabling cell corpse clearance in Caenorhabditis elegans. Here, we found that the C. elegans intracellular protein sorting complex, retromer, was required for cell corpse clearance by mediating the recycling of CED-1. Retromer was recruited to the surfaces of phagosomes containing cell corpses, and its loss of function caused defective cell corpse removal. The retromer probably acted through direct interaction with CED-1 in the cell corpse recognition pathway. In the absence of retromer function, CED-1 associated with lysosomes and failed to recycle from phagosomes and cytosol to the plasma membrane. Thus, retromer is an essential mediator of apoptotic cell clearance by regulating phagocytic receptor(s) during cell corpse engulfment.
In many cancer types, integrin-mediated signaling regulates proliferation, survival and invasion of tumorigenic cells. However, it is still unclear how integrins crosstalk with oncogenes to regulate tumorigenesis and metastasis. Here we show that oncogenic K-RasV12 upregulates α6-integrin expression in Madin–Darby canine kidney (MDCK) cells via activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)/Fos-related antigen 1-signaling cascade. Activated α6-integrins promoted metastatic capacity and anoikis resistance, and led to perturbed growth of MDCK cysts. Transcriptomic analysis of K-RasV12-transformed MDCK cells also revealed robust downregulation of αV-class integrins. Re-expression of αV-integrin in K-RasV12-transformed MDCK cells synergistically upregulated the expression of Zinc finger E-box-binding homeobox 1 and Twist-related protein 1 and triggered epithelial-mesenchymal transition leading to induced cell motility and invasion. These results delineate the signaling cascades connecting oncogenic K-RasV12 with α6- and αV-integrin functions to modulate cancer cell survival and tumorigenesis, and reveal new possible strategies to target highly oncogenic K-RasV12 mutants.
Cells in tissues in vivo face a very different microenvironment than typical cultured cells plated on a plastic dish. Already several decades ago, cell biologists observed that cell lines show dramatically different morphology and growth characteristics when embedded into 3-dimensional (3D) substrates or standard tissue culture plates (Barcellos-Hoff et al. 1989, Montesano et al. 1991, Simian and Bissell 2017. Despite its imminent benefit for cell biological studies, suspicion and prejudice towards more complicated sample preparation requirements limited the popularity of 3D culture techniques until recently, when it was shown that soft 3D gels made of basement membrane extracts (BME) allow prolonged culture of many types of primary epithelial cells (Clevers 2016, Sato et al. 2009). These observations have brought 3D organoid culture systems into the mainstream. Here we describe two protocols for culturing epithelial cells in 3D substrates, the "blob culture" setup where cells are fully embedded into BME gel and the "overlay setup" where cells are seeded on top of BME gel and then overlaid with a thin layer of BME (Bryant et al. 2010, Debnath andBrugge 2005).
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