NHA2 promotes cyst development in an in vitro model of polycystic kidney disease.

KEY POINTS: Significant and selective up-regulation of the Na+ /H+ exchanger NHA2 (SLC9B2) was observed in cysts of patients with autosomal dominant polycystic kidney disease. Using the MDCK cell model of cystogenesis, it was found that NHA2 increases cyst size. Silencing or pharmacological inhibition of NHA2 inhibits cyst formation in vitro. Polycystin-1 represses NHA2 expression via Ca2+ /NFAT signalling whereas the dominant negative membrane-anchored C-terminal fragment (PC1-MAT) increased NHA2 levels. Drugs (caffeine, theophylline) and hormones (vasopressin, aldosterone) known to exacerbate cysts elicit NHA2 expression. Taken together, the findings reveal NHA2 as a potential new player in salt and water homeostasis in the kidney and in the pathogenesis of polycystic kidney disease. ABSTRACT: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2 encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The molecular pathways linking polycystins to cyst development in ADPKD are still unclear. Intracystic fluid secretion via ion transporters and channels plays a crucial role in cyst expansion in ADPKD. Unexpectedly, we observed significant and selective up-regulation of NHA2, a member of the SLC9B family of Na+ /H+ exchangers, that correlated with cyst size and disease severity in ADPKD patients. Using three-dimensional cultures of MDCK cells to model cystogenesis in vitro, we showed that ectopic expression of NHA2 is causal to increased cyst size. Induction of PC1 in MDCK cells inhibited NHA2 expression with concordant inhibition of Ca2+ influx through store-dependent and -independent pathways, whereas reciprocal activation of Ca2+ influx by the dominant negative membrane-anchored C-terminal tail fragment of PC1 elevated NHA2. We showed that NHA2 is a target of Ca2+ /NFAT signalling and is transcriptionally induced by methylxanthine drugs such as caffeine and theophylline, which are contraindicated in ADPKD patients. Finally, we observed robust induction of NHA2 by vasopressin, which is physiologically consistent with increased levels of circulating vasopressin and up-regulation of vasopressin V2 receptors in ADPKD. Our findings have mechanistic implications on the emerging use of vasopressin V2 receptor antagonists such as tolvaptan as safe and effective therapy for polycystic kidney disease and reveal a potential new regulator of transepithelial salt and water transport in the kidney.

Circulating tumor DNA: current challenges for clinical utility.

Cancer cells shed naked DNA molecules into the circulation. This circulating tumor DNA (ctDNA) has become the predominant analyte for liquid biopsies to understand the mutational landscape of cancer. Coupled with next-generation sequencing, ctDNA can serve as an alternative substrate to tumor tissues for mutation detection and companion diagnostic purposes. In fact, recent advances in precision medicine have rapidly enabled the use of ctDNA to guide treatment decisions for predicting response and resistance to targeted therapies and immunotherapies. An advantage of using ctDNA over conventional tissue biopsies is the relatively noninvasive approach of obtaining peripheral blood, allowing for simple repeated and serial assessments. Most current clinical practice using ctDNA has endeavored to identify druggable and resistance mutations for guiding systemic therapy decisions, albeit mostly in metastatic disease. However, newer research is evaluating potential for ctDNA as a marker of minimal residual disease in the curative setting and as a useful screening tool to detect cancer in the general population. Here we review the history of ctDNA and liquid biopsies, technologies to detect ctDNA, and some of the current challenges and limitations in using ctDNA as a marker of minimal residual disease and as a general blood-based cancer screening tool. We also discuss the need to develop rigorous clinical studies to prove the clinical utility of ctDNA for future applications in oncology.

Secretory pathway Ca2+-ATPase SPCA2 regulates mitochondrial respiration and DNA damage response through store-independent calcium entry.

A complex interplay between the extracellular space, cytoplasm and individual organelles modulates Ca2+ signaling to impact all aspects of cell fate and function. In recent years, the molecular machinery linking endoplasmic reticulum stores to plasma membrane Ca2+ entry has been defined. However, the mechanism and pathophysiological relevance of store-independent modes of Ca2+ entry remain poorly understood. Here, we describe how the secretory pathway Ca2+-ATPase SPCA2 promotes cell cycle progression and survival by activating store-independent Ca2+ entry through plasma membrane Orai1 channels in mammary epithelial cells. Silencing SPCA2 expression or briefly removing extracellular Ca2+ increased mitochondrial ROS production, DNA damage and activation of the ATM/ATR-p53 axis leading to G0/G1 phase cell cycle arrest and apoptosis. Consistent with these findings, SPCA2 knockdown confers redox stress and chemosensitivity to DNA damaging agents. Unexpectedly, SPCA2-mediated Ca2+ entry into mitochondria is required for optimal cellular respiration and the generation of mitochondrial membrane potential. In hormone receptor positive (ER+/PR+) breast cancer subtypes, SPCA2 levels are high and correlate with poor survival prognosis. We suggest that elevated SPCA2 expression could drive pro-survival and chemotherapy resistance in cancer cells, and drugs that target store-independent Ca2+ entry pathways may have therapeutic potential in treating cancer.

An integrated functional and clinical genomics approach reveals genes driving aggressive metastatic prostate cancer.

Genomic sequencing of thousands of tumors has revealed many genes associated with specific types of cancer. Similarly, large scale CRISPR functional genomics efforts have mapped genes required for cancer cell proliferation or survival in hundreds of cell lines. Despite this, for specific disease subtypes, such as metastatic prostate cancer, there are likely a number of undiscovered tumor specific driver genes that may represent potential drug targets. To identify such genetic dependencies, we performed genome-scale CRISPRi screens in metastatic prostate cancer models. We then created a pipeline in which we integrated pan-cancer functional genomics data with our metastatic prostate cancer functional and clinical genomics data to identify genes that can drive aggressive prostate cancer phenotypes. Our integrative analysis of these data reveals known prostate cancer specific driver genes, such as AR and HOXB13, as well as a number of top hits that are poorly characterized. In this study we highlight the strength of an integrated clinical and functional genomics pipeline and focus on two top hit genes, KIF4A and WDR62. We demonstrate that both KIF4A and WDR62 drive aggressive prostate cancer phenotypes in vitro and in vivo in multiple models, irrespective of AR-status, and are also associated with poor patient outcome.

A Ca2+-ATPase Regulates E-cadherin Biogenesis and Epithelial-Mesenchymal Transition in Breast Cancer Cells.

Progression of benign tumors to invasive, metastatic cancer is accompanied by the epithelial-to-mesenchymal transition (EMT), characterized by loss of the cell-adhesion protein E-cadherin. Although silencing mutations and transcriptional repression of the E-cadherin gene have been widely studied, not much is known about posttranslational regulation of E-cadherin in tumors. We show that E-cadherin is tightly coexpressed with the secretory pathway Ca2+-ATPase isoform 2, SPCA2 (ATP2C2), in breast tumors. Loss of SPCA2 impairs surface expression of E-cadherin and elicits mesenchymal gene expression through disruption of cell adhesion in tumorspheres and downstream Hippo-YAP signaling. Conversely, ectopic expression of SPCA2 in triple-negative breast cancer elevates baseline Ca2+ and YAP phosphorylation, enhances posttranslational expression of E-cadherin, and suppresses mesenchymal gene expression. Thus, loss of SPCA2 phenocopies loss of E-cadherin in the Hippo signaling pathway and EMT-MET transitions, consistent with a functional role for SPCA2 in E-cadherin biogenesis. Furthermore, we show that SPCA2 suppresses invasive phenotypes, including cell migration in vitro and tumor metastasis in vivo. Based on these findings, we propose that SPCA2 functions as a key regulator of EMT and may be a potential therapeutic target for treatment of metastatic cancer. IMPLICATIONS: Posttranslational control of E-cadherin and the Hippo pathway by calcium signaling regulates EMT in breast cancer cells.