Faculty Bibliography

Mutations in the polycystins cause autosomal dominant polycystic kidney disease (ADPKD). Here we show that transmembrane protein 33 (TMEM33) interacts with the ion channel polycystin-2 (PC2) at the endoplasmic reticulum (ER) membrane, enhancing its opening over the whole physiological calcium range in ER liposomes fused to planar bilayers. Consequently, TMEM33 reduces intracellular calcium content in a PC2-dependent manner, impairs lysosomal calcium refilling, causes cathepsins translocation, inhibition of autophagic flux upon ER stress, as well as sensitization to apoptosis. Invalidation of TMEM33 in the mouse exerts a potent protection against renal ER stress. By contrast, TMEM33 does not influence pkd2-dependent renal cystogenesis in the zebrafish. Together, our results identify a key role for TMEM33 in the regulation of intracellular calcium homeostasis of renal proximal convoluted tubule cells and establish a causal link between TMEM33 and acute kidney injury.

Increased levels of the calcium-binding protein neuronal calcium sensor 1 (NCS1) predict an unfavorable patient outcome in several aggressive cancers, including breast and liver tumors. Previous studies suggest that NCS1 overexpression facilitates metastatic spread of these cancers. To investigate this hypothesis, we explored the effects of NCS1 overexpression on cell proliferation, survival, and migration patterns in vitro in 2- and 3-dimensional (2/3-D). Furthermore, we translated our results into an in vivo mouse xenograft model. Cell-based proliferation assays were used to demonstrate the effects of overexpression of NCS1 on growth rates. In vitro colony formation and wound healing experiments were performed and 3-D migration dynamics were studied using collagen gels. Nude mice were injected with breast cancer cells to monitor NCS1-dependent metastasis formation over time. We observed that increased NCS1 levels do not change cellular growth rates, but do significantly increase 2- and 3-D migration dynamics in vitro. Likewise, NCS1-overexpressing cells have an increased capacity to form distant metastases and demonstrate better survival and less necrosis in vivo. We found that NCS1 preferentially localizes to the leading edge of cells and overexpression increases the motility of cancer cells. Furthermore, this phenotype is correlated with an increased number of metastases in a xenograft model. These results lay the foundation for exploring the relevance of an NCS1-mediated pathway as a metastatic biomarker and as a target for pharmacologic interventions.-Apasu, J. E., Schuette, D., LaRanger, R., Steinle, J. A., Nguyen, L. D., Grosshans, H. K., Zhang, M., Cai, W. L., Yan, Q., Robert, M. E., Mak, M., Ehrlich, B. E. Neuronal calcium sensor 1 (NCS1) promotes motility and metastatic spread of breast cancer cells in vitro and in vivo.

Neuronal Calcium Sensor-1 (NCS-1) is a highly conserved calcium binding protein which contributes to the maintenance of intracellular calcium homeostasis and regulation of calcium-dependent signaling pathways. It is involved in a variety of physiological cell functions, including exocytosis, regulation of calcium permeable channels, neuroplasticity and response to neuronal damage. Over the past 30 years, continuing investigation of cellular functions of NCS-1 and associated disease states have highlighted its function in the pathophysiology of several disorders and as a therapeutic target. Among the diseases that were found to be associated with NCS-1 are neurological disorders such as bipolar disease and non-neurological conditions such as breast cancer. Furthermore, alteration of NCS-1 expression is associated with substance abuse disorders and severe side effects of chemotherapeutic agents. The objective of this article is to summarize the current body of evidence describing NCS-1 and its interactions on a molecular and cellular scale, as well as describing macroscopic implications in physiology and medicine. Particular attention is paid to the role of NCS-1 in development and prevention of chemotherapy induced peripheral neuropathy (CIPN).

Peripheral neuropathy is a major adverse effect in the use of chemotherapeutic drugs. In nearly 50% of patients, chemotherapy induced peripheral neuropathy (CIPN) has been reported as irreversible. With increasing numbers of patients surviving treatment as well as increasing duration of survival after treatment, reducing the side effects of chemotherapy and improving the quality of life has become a major focus of cancer survivorship. Multiple classes of chemotherapeutic drugs including taxanes, platinum agents and vinka alkaloids list peripheral neuropathy as the main dose-limiting side effect of treatment. We previously found that drugs that interfere with the microtubule function, including taxanes and vinca alkaloids, bind to neuronal calcium sensor 1 (NCS1), leading to aberrant calcium signaling. The altered calcium signaling can be mitigated by application of drugs used to treat bipolar disease (e.g., lithium and valproic acid) prior to initiation of chemotherapy. Because pre-treatment with these drugs prevented CIPN in mice treated with taxanes, we sought clinical evidence by performing a retrospective chart review study of the VA electronic health record to see whether or not there would be evidence to support our scientific belief that patients treated with lithium or valproic acid while receiving chemotherapy have a lower risk for development of CIPN than patients who received chemotherapy alone. Our data did provide evidence supporting the belief that treatment with lithium or valproic acid concurrently with chemotherapy was associated with a decreased incidence of developing CIPN.

Mutations in polycystin-1 (PC1) and polycystin-2 (PC2) result in a commonly occurring genetic disorder, called Autosomal Dominant Polycystic Kidney Disease (ADPKD), that is characterized by the formation and development of kidney cysts. Epithelial cells with loss-of-function of PC1 or PC2 show higher rates of proliferation and apoptosis and reduced autophagy. PC1 is a large multifunctional transmembrane protein that serves as a sensor that is usually found in complex with PC2, a calcium (Ca²⁺)-permeable cation channel. In addition to decreased Ca²⁺ signaling, several other cell fate-related pathways are de-regulated in ADPKD, including cAMP, MAPK, Wnt, JAK-STAT, Hippo, Src, and mTOR. In this review we discuss how polycystins regulate cell death and survival, highlighting the complexity of molecular cascades that are involved in ADPKD.

Neuronal calcium sensor-1 (NCS-1) has been identified as a binding partner of the taxane, paclitaxel. Our previous study showed that overexpression of NCS-1 increased the efficacy of paclitaxel in vitro, but was associated with poor clinical outcome. Here, we determine if NCS-1 expression is associated with pathological complete response (pCR) to taxane-based neoadjuvant chemotherapy in 105 pre-treatment breast cancer biopsies. Elevated expression of NCS-1 was found to be positively associated with pCR. These results suggest that NCS-1 may be a predictive biomarker for response to taxane-based neoadjuvant chemotherapy in breast cancer.

Although autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple kidney cysts, the most frequent cause of death in ADPKD patients is cardiovascular disease. ADPKD is linked to mutations in PKD1 or pkd2, the genes that encode for the proteins polycystin 1 and polycystin 2 (PC1 and PC2, respectively). The cardiovascular complications have been assumed to be a consequence of renal hypertension and activation of renin/angiotensin/aldosterone (RAAS) pathway. However, the expression of PC1 and PC2 in cardiac tissue suggests additional direct effects of these proteins on cardiac function. We previously reported that zebrafish lacking PC2 develop heart failure, and that heterozygous Pkd2^+/- mice are hypersensitive to acute β-adrenergic receptor (βAR) stimulation. Here, we investigate the effect of cardiac stress (prolonged continuous βAR stimulus) on Pkd2^+/- mice. After βAR stimulation for 7 days, wild-type (WT) mice had increased left ventricular mass and natriuretic peptide (ANP and BNP) mRNA levels. The WT mice also had upregulated levels of PC2 and chromogranin B (CGB, an upstream regulator of BNP). Conversely, Pkd2^+/- mice had increased left ventricular mass, but natriuretic peptide and CGB expression levels remained constant. Reversal of the increased cardiac mass was observed in WT mice 3 days after cessation of the βAR stimulation, but not in Pkd2^+/- mice. We suggest that cardiac stress leads to upregulation of the PC2-CGB-BNP signaling axis, and this pathway regulates the production of cardio-protective natriuretic peptides. The lack of a PC2-dependent cardio-protective function may contribute to the severity of cardiac dysfunction in Pkd2^+/- mice and in ADPKD patients.

Starvation induces liver autophagy, which is thought to provide nutrients for use by other organs and thereby maintain whole-body homeostasis. Here we demonstrate that O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is required for glucagon-stimulated liver autophagy and metabolic adaptation to starvation. Genetic ablation of OGT in mouse livers reduces autophagic flux and the production of glucose and ketone bodies. Upon glucagon-induced calcium signaling, calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates OGT, which in turn promotes O-GlcNAc modification and activation of Ulk proteins by potentiating AMPK-dependent phosphorylation. These findings uncover a signaling cascade by which starvation promotes autophagy through OGT phosphorylation and establish the importance of O-GlcNAc signaling in coupling liver autophagy to nutrient homeostasis.

Neuronal Calcium Sensor 1 (NCS-1) is a multi-functional Ca²⁺-binding protein that affects a range of cellular processes beyond those related to neurons. Functional characterization of NCS-1 in neuronal model systems suggests that NCS-1 may influence oncogenic processes. To this end, the biological role of NCS-1 was investigated by altering its endogenous expression in MCF-7 and MB-231 breast cancer cells. Overexpression of NCS-1 resulted in a more aggressive tumor phenotype demonstrated by a marked increase in invasion and motility, and a decrease in cell-matrix adhesion to collagen IV. Overexpression of NCS-1 was also shown to increase the efficacy of paclitaxel-induced cell death in a manner that was independent of cellular proliferation. To determine the association between NCS-1 and clinical outcome, NCS-1 expression was measured in two independent breast cancer cohorts by the Automated Quantitative Analysis method of quantitative immunofluorescence. Elevated levels of NCS-1 were significantly correlated with shorter survival rates. Furthermore, multivariate analysis demonstrated that NCS-1 status was prognostic, independent of estrogen receptor, progesterone receptor, HER2, and lymph node status. These findings indicate that NCS-1 plays a role in the aggressive behavior of a subset of breast cancers and has therapeutic or biomarker potential.Implications: NCS-1, a calcium-binding protein, is associated with clinicopathologic features of aggressiveness in breast cancer cells and worse outcome in two breast cancer patient cohorts. Mol Cancer Res; 15(7); 942-52. ©2017 AACR.