Do you know the sex of your cells? Not a question that is frequently heard around the lab bench, yet thanks to recent research is probably one that should be asked. It is self-evident that cervical epithelial cells would be derived from female tissue and prostate cells from a male subject (exemplified by HeLa and LnCaP, respectively), yet beyond these obvious examples, it would be true to say that the sex of cell lines derived from non-reproductive tissue, such as lung, intestine, kidney, for example, is given minimal if any thought. After all, what possible impact could the presence of a Y chromosome have on the biochemistry and cell biology of tissues such as the exocrine pancreatic acini? Intriguingly, recent evidence has suggested that far from being irrelevant, genes expressed on the sex chromosomes can have a marked impact on the biology of such diverse tissues as neurons and renal cells. It is also policy of AJP-Cell Physiology that the source of all cells utilized (species, sex, etc.) should be clearly indicated when submitting an article for publication, an instruction that is rarely followed (http://www.the-aps.org/mm/Publications/Info-For-Authors/Composition). In this review we discuss recent data arguing that the sex of cells being used in experiments can impact the cell's biology, and we provide a table outlining the sex of cell lines that have appeared in AJP-Cell Physiology over the past decade.

Mcl-1 is an antiapoptotic member of the Bcl-2 family frequently upregulated in non-small cell lung carcinoma (NSCLC). We now report the physiological significance of an interaction between Mcl-1 and the mitochondrial outer membrane-localized voltage-dependent anion channel (VDAC) in NSCLC cell lines. Mcl-1 bound with high affinity to VDAC1 and 3 isoforms but only very weakly to VDAC2 and binding was disrupted by peptides based on the VDAC1 sequence. In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca2+ uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation. In A549, H1299 and H460 cells, both Mcl-1 knockdown and VDAC-based peptides attenuated cell migration without affecting cell proliferation. Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration. These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca2+-dependent ROS production.

Progression from early forms of prostate cancer to castration-resistant disease is associated with an increase in signal transduction activity. The majority of castration-resistance cancers persist in the expression of the androgen receptor (AR), as well as androgen-dependent genes. The AR is regulated not only by it associated steroid hormone, but also by manifold regulatory and signaling molecules, including several kinases. We undertook evaluation of the role of Lemur Tyrosine Kinase 2 (LMTK2) in modulating AR activity, as several Genome Wide Association Studies (GWAS) have shown a marked association of LMTK2 activity with the development of prostate cancer. We confirm that not only is LMTK2 mRNA reduced in prostate cancer tissue, but also LMTK2 protein levels are markedly diminished. Knockdown of LMTK2 protein in prostate cell lines greatly increased the transcription of androgen-responsive genes. In addition, LMTK2 knockdown led to an increase in prostate cancer stem cell populations in LNCaP cells, indicative of increased tumorogenicity. Using multiple approaches, we also demonstrate that LMTK2 interacts with the AR, thus putting LMTK2 as a component of a signaling complex modulating AR activity. Our finding that LMTK2 is a negative regulator of AR activity defines a novel cellular pathway for activation of AR-responsive genes in castrate resistant-prostate cancer. Moreover, pharmacologic manipulation of LMTK2 activity will provide a novel therapeutic target for more effective treatments for patients with castrate-resistant prostate cancer.

Lemur tyrosine kinase 3 (LMTK3) is an oncogenic kinase that is involved in different types of cancer (breast, lung, gastric, colorectal) and biological processes including proliferation, invasion, migration, chromatin remodeling as well as innate and acquired endocrine resistance. However, the role of LMTK3 in response to cytotoxic chemotherapy has not been investigated thus far. Using both 2D and 3D tissue culture models, we found that overexpression of LMTK3 decreased the sensitivity of breast cancer cell lines to cytotoxic (doxorubicin) treatment. In a mouse model we showed that ectopic overexpression of LMTK3 decreases the efficacy of doxorubicin in reducing tumor growth. Interestingly, breast cancer cells overexpressing LMTK3 delayed the generation of double strand breaks (DSBs) after exposure to doxorubicin, as measured by the formation of γH2AX foci. This effect was at least partly mediated by decreased activity of ataxia-telangiectasia mutated kinase (ATM) as indicated by its reduced phosphorylation levels. In addition, our RNA-seq analyses showed that doxorubicin differentially regulated the expression of over 700 genes depending on LMTK3 protein expression levels. Furthermore, these genes were found to promote DNA repair, cell viability and tumorigenesis processes / pathways in LMTK3-overexpressing MCF7 cells. In human cancers, immunohistochemistry staining of LMTK3 in pre- and post-chemotherapy breast tumor pairs from four separate clinical cohorts revealed a significant increase of LMTK3 following both doxorubicin and docetaxel based chemotherapy. In aggregate, our findings show for the first time a contribution of LMTK3 in cytotoxic drug resistance in breast cancer.

Androgen receptor (AR), is a transcription factor and a member of a hormone receptor superfamily. AR plays a vital role in the progression of prostate cancer and is a crucial target for therapeutic interventions. While the majority of advanced-stage prostate cancer patients will initially respond to the androgen deprivation, the disease often progresses to castrate-resistant prostate cancer (CRPC). Interestingly, CRPC tumors continue to depend on hyperactive AR signaling and will respond to potent second-line antiandrogen therapies, including bicalutamide (CASODEX®) and enzalutamide (XTANDI®). However, the progression-free survival rate for the CRPC patients on antiandrogen therapies is only 8–19 months. Hence, there is a need to understand the mechanisms underlying CRPC progression and eventual treatment resistance. Here, we have leveraged next-generation sequencing and newly developed analytical methodologies to evaluate the role of AR signaling in regulating the transcriptome of prostate cancer cells. The genomic and pharmacologic stimulation and inhibition of AR activity demonstrates that AR regulates alternative splicing within cancer-relevant genes. Furthermore, by integrating transcriptomic data from in vitro experiments and in prostate cancer patients, we found that a significant number of AR-regulated splicing events are associated with tumor progression. For example, we found evidence for an inadvertent AR-antagonist-mediated switch in IDH1 and PL2G2A isoform expression, which is associated with a decrease in overall survival of patients. Mechanistically, we discovered that the epithelial-specific splicing regulators (ESRP1 and ESRP2), flank many AR-regulated alternatively spliced exons. And, using 2D invasion assays, we show that the inhibition of ESRPs can suppress AR-antagonist-driven tumor invasion. Our work provides evidence for a new mechanism by which AR alters the transcriptome of prostate cancer cells by modulating alternative splicing. As such, our work has important implications for CRPC progression and development of resistance to treatment with bicalutamide and enzalutamide.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel composed of 1480 amino acids. The major mutation responsible for cystic fibrosis results in loss of amino acid residue, F508, (F508del). Loss of F508 in CFTR alters the folding pathway resulting in endoplasmic reticulum associated degradation (ERAD). This study investigates the role of synonymous codon in the expression of CFTR and CFTR F508del in human HEK293 cells. DNA encoding the open reading frame (ORF) for CFTR containing synonymous codon replacements, were expressed using a heterologous vector integrated into the genome. The results indicate that the codon usage greatly affects the expression of CFTR. While the promoter strength driving expression of the ORFs was largely unchanged and the mRNA half-lives were unchanged, the steady state levels of the mRNA varied by as much as 30 fold. Experiments support that this apparent inconsistency is attributed to exon junction complex independent nonsense mediated decay. The ratio of CFTR/mRNA indicates that mRNA containing native codons was more efficient in expressing mature CFTR as compared to mRNA containing synonymous high expression codons. However, when F508del CFTR was expressed after codon optimization, a greater percentage of the protein escaped ERAD resulting in considerable levels of mature F508del CFTR on the plasma membrane, which showed channel activity. These results indicate that for CFTR, codon usage has an effect on mRNA levels, protein expression and likely, for F508del CFTR, chaperone assisted folding pathway.

The recent FDA approval of two drugs to treat the basic defect in cystic fibrosis has given hope to patients and their families battling this devastating disease. Over many years, with heavy financial investment from Vertex Pharmaceuticals and the Cystic Fibrosis Foundation, pre-clinical evaluation of thousands of synthetic drugs resulted in the production of Kalydeco and Orkambi. Yet, despite the success of this endeavor, many other compounds have been proposed as therapeutic agents in the treatment of CF. Of note, several of these compounds are naturally occurring, and are present in spices from the grocery store and over the counter preparations in health food stores. In this short review, we look at three such compounds, genistein, curcumin, and resveratrol, and evaluate the scientific support for their use as therapeutic agents in the treatment of patients with CF.

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