Expression of the voltage gated proton channel (H_v1) as identified by immunocytochemistry has been reported previously in breast cancer tissue. Increased expression of H_V1 was correlated with poor prognosis and decreased overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of H_V1 channel activity, confirming its presence and function in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we identify significant levels of H_V1 expression in 3 out of 8 “triple negative” breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of H_V1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of H_V1 using shRNA (knock-down; KD) and by eliminating H_V1 using CRISPR/Cas9 gene editing (knock-out; KO). Surprisingly, these two approaches produced incongruous effects. Knock-down of H_V1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H₂O₂ release. In contrast, H_V1 Knock-out cells did not show reduced migration or H₂O₂ release. H_V1 KO but not KD cells showed an increased glycolytic rate accompanied by an increase in p-AKT (phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in H_V1 KO cells. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from H_V1 KO cells grew less than WT in mass, with lower staining for the Ki-67 marker for cell proliferation rate. Therefore, deletion of H_V1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus appears to be independent of oxidant production by NADPH oxidase molecules and to be mediated by cell adhesion molecules. Although H_V1 KO and KD affect certain cellular mechanisms differently, both implicate H_V1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.

The voltage-gated proton channel (H_V1) is a voltage sensor that also conducts protons. The singular ability of protons to penetrate proteins complicates distinguishing closed and open channels. When we replaced valine with histidine at position 116 in the external vestibule of hH_V1, current was potently inhibited by externally applied Zn²⁺ in a construct lacking the two His that bind Zn²⁺ in WT channels. High-affinity binding with profound effects at 10 nM Zn²⁺ at pH_o 7 suggests additional groups contribute. We hypothesized that Asp¹⁸⁵, which faces position 116 in our closed-state model, contributes to Zn²⁺ chelation. Confirming this prediction, V116H/D185N abolished Zn²⁺ binding. Studied in a C-terminal truncated monomeric construct, V116H channels activated rapidly. Anomalously, Zn²⁺ slowed activation, producing a time constant independent of both voltage and Zn²⁺ concentration. We hypothesized that slow turn-on of H⁺ current in the presence of Zn²⁺ reflects the rate of Zn²⁺ unbinding from the channel, analogous to drug-receptor dissociation reactions. This behavior in turn suggests that the affinity for Zn²⁺ is greater in the closed state of hH_V1. Supporting this hypothesis, pulse pairs revealed a rapid component of activation whose amplitude decreased after longer intervals at negative voltages as closed channels bound Zn²⁺. The lower affinity of Zn²⁺ in open channels is consistent with the idea that structural rearrangements within the transmembrane region bring Arg²⁰⁵ near position 116, electrostatically expelling Zn²⁺. This phenomenon provides direct evidence that Asp¹⁸⁵ opposes position 116 in closed channels and that Arg²⁰⁵ moves between them when the channel opens.

Head and neck cancer (HNC) is a challenging disease that lacks effective treatment, particularly in the cases that spread locoregionally and metastasize distantly, dramatically reducing patient survival rates. Expanding the understanding of the mechanisms of the metastatic cascade is critical for creating more effective therapeutics that improve outcomes for HNC patients. A true grasp of cancer metastasis requires the consideration of all cell types that contribute to the inflammatory HNC microenvironment as drivers of this process. More emphasis now is being placed on exploring the roles of the different immune cells in cancer control, tumorigenesis and metastasis. Myeloid cells are the most numerous immune cell types in the body, and they are actively recruited and reprogrammed by tumor cells to behave in a variety of ways. These cells are remarkably diverse in phenotype and function, and the part they play in tumor spread greatly differs based on the cell type. This review will focus on summarizing the roles of macrophages, neutrophils, myeloid derived suppressor cells (MDSCs), and dendritic cells (DCs) in driving HNC metastasis by examining the current knowledge base and offering potential new routes through which to target and treat this deadly process.

Recurrence in early-stage laryngeal carcinoma after radiation therapy often necessitates a total salvage laryngectomy (TSL). Understanding the genetic landscape that influences cancer progression after radiation therapy may help future treatments. We investigated early-stage laryngeal carcinoma patients treated with radiotherapy to identify mutation patterns that predispose to disease recurrence. The patient cohort had 35 early-stage laryngeal head and neck squamous cell carcinoma (HNSCC) patients (T1 = 15, T2 = 20) treated with radiotherapy (RT). We stratified patients: Responders (no recurrence, N = 14), non-responders (recurrence within 12 months, N = 21), and non-responders undergoing TSL (N = 18). We employed whole exome sequencing to characterize gene mutations using the Genome Analysis Toolkit (GATK) for variant detection and impact on critical biological pathways and post-radiotherapy patient outcomes. Pathway analysis Ingenuity and Reactome Pathway Analysis tools were used to explore the mutated gene pathways. Differential mutation analysis was performed in the respective groups to find driver genes. In the pre-treatment samples, KCNT2 and AGAP6 mutations were exclusively found in non-responders (OR = 0, P = 0.005 and OR = 0, P = 0.027, respectively), while ADAMTS7 mutations were solely present in responders (OR = inf, P = 0.019). PLEC mutations were more prevalent in responders (OR = 11.6, P = 0.006). Pathway analysis revealed that significant genes were involved in the RND2 GTPase cycle, protein O-glycosylation-related diseases, and apoptotic pathways. Post-treatment analysis in patients undergoing TSL had enrichment of mutations in apoptosis regulation pathways. The study reveals that mutations in apoptosis controlling genes were predominantly represented in the larynx non-responder patients both in the pre-radiotherapy and post-TSL populations.

Expression of the voltage gated proton channel (H v 1) as identified by immunocytochemistry has been previously reported in breast cancer tissue. Increased expression of H V 1 was correlated with poor prognosis and decreases overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of H V 1 channel activity thus, demonstrating their presence and functional properties in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we also identify significant levels of H V 1 expression in 3 out of 8 “triple negative” breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of H V 1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of H V 1 using shRNA (“knock-down;”“KD”) and by eliminating H V 1 using CRISPR/Cas9 gene editing (“knock-out”; “KO”). However, these two approaches produced different effects. Knock-down of H V 1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H 2 O 2 release. In contrast, H V 1 KO cells do not show reduction in migration or H 2 O 2 release. H V 1 KO but not knock-down cells showed an increased glycolytic rate with an accompanied increase in p-AKT (Phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in the absence of H V 1 expression. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from H V 1 KO cells grew less in mass with lower staining for the Ki-67 maker for cell proliferation rate. Therefore, deletion of H V 1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus, appears to be independent of oxidant production by NADPH oxidase molecules and be determined through cell adhesion activity. While H V 1 KO results in cell mechanisms different from KD, both implicate H V 1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.