Clinicopathological and biological significance of human voltage-gated proton channel Hv1 protein overexpression in breast cancer

In our previous work, we showed for the first time that the voltage-gated proton channel Hv1 is specifically expressed in highly metastatic human breast tumor tissues and cell lines. However, the contribution of Hv1 to breast carcinogenesis is not well known. In this study, we showed that Hv1 expression was significantly correlated with the tumor size (p = 0.001), tumor classification (p = 0.000), lymph node status (p = 0.000), clinical stage (p = 0.000), and Her-2 status (p = 0.045). High Hv1 expression was associated significantly with shorter overall (p = 0.000) and recurrence-free survival (p = 0.000). In vitro, knockdown of Hv1 expression in the highly metastatic MDA-MB-231 cells decreased the cell proliferation and invasiveness, inhibited the cell proton secretion and intracellular pH recovery, and blocked the cell capacity of acidifying extracellular milieu. Furthermore, the gelatinase activity in MDA-MB-231 cells that suppressed Hv1 was reduced. In vivo, the breast tumor size of the implantation of the MDA-MB-231 xenografts in nude mice that were knocked down by Hv1 was dramatically smaller than that in the control groups. The results demonstrated that the inhibition of Hv1 function via knockdown of Hv1 expression can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity. Based on these results, we came to the conclusion that Hv1 is a potential biomarker for prognosis of breast cancer and a potential target for anticancer drugs in breast cancer therapy.     

Zn induces apoptosis in human highly metastatic SHG-44 glioma cells,through inhibiting activity of the voltage-gated proton channel Hv1

In contrast to the voltage-gated K⁺ channels, the voltage-gated proton channel Hv1 contains a voltage-sensor domain but lacks a pore domain. Here, we showed that Hv1 is expressed in the highly metastatic glioma cell SHG-44, but lowly in the poorly metastatic glioma cell U-251. Inhibition of Hv1 activity by 140 μM zinc chloride induces apoptosis in the human highly metastatic glioma cells. Zn²⁺ ions markedly inhibit proton secretion, and reduce the gelatinase activity in the highly metastatic glioma cells. In vivo, the glioma tumor sizes of the implantation of the SHG-44 xenografts in nude mice that were injected zinc chloride solution, were dramatically smaller than that in the controlled groups. The results demonstrated that the inhibition of Hv1 activity via Zn²⁺ ions can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity. Our results suggest that Zn²⁺ ions may be used as a potential anti-glioma drug for glioma therapy.     

Enhanced expression of glucose transporter GLUT3 in tumorigenic HeLa cell hybrids associated with tumor suppressor dysfunction.

Previous studies on human cell hybrids between HeLa and normal human fibroblasts have indicated that the tumorigenicy may be controlled by a putative tumor suppressor gene on chromosome 11. We previously demonstrated a twofold increase in glucose uptake with a reduced Km by tumorigenic HeLa cell hybrids which expressed a highly glycosylated GLUT1. In this study, we reported that a tumorigenic cell hybrid, CGL4, also expressed a glucose transporter isoform, GLUT3, that was undetectable in nontumorigenic CGL1 cells. The expression of GLUT3 together with GLUT1 of 70 kDa was also evident in three gamma-ray-induced tumorigenic clones isolated from CGL1 cells, while control nontumorigenic irradiated cells expressed 50 kDa GLUT1 alone. In accordance with this, GLUT3 mRNA was specifically expressed in tumorigenic cell hybrids. To examine the role of GLUT3, clones which stably overexpress GLUT3 were developed from both CGL1 and CGL4 cells. In these transfectants, the affinity for 2-deoxyglucose markedly increased, in parallel with the amount of expressed GLUT3 irrespective of its N-glycosylation state. These results suggest that the enhanced GLUT3 expression in HeLa cell hybrids associated with the tumorigenic phenotypes may account for the increased affinity for 2-deoxyglucose. Possible roles of the putative tumor suppressor in control of gene expression and glucose uptake is discussed.     

Nuclear control of tumorigenicity in cells reconstructed by PEG-induced fusion of cell fragments

The techniques of somatic cell hybridization have provided a valuable means of studying mechanisms of regulation of mammalian cell differentiation and transformation. Most previous studies have indicated that fusions between tumorigenic and nontumorigenic cells result in hybrid cells that are usually tumorigenic. In recent years it has been demonstrated that the phenotypic expression of tumorigenicity is at least partially due to the extensive chromosome loss that occurs in most interspecific and some intraspecific hybrid cells. In the present study we have utilized enucleation techniques that permit cells to be divided into nuclear (karyoplast) and cytoplasmic (cytoplast) cell fragments. Even though these nuclear and cytoplasmic fragments are metabolically stable for short periods of time, in our hands they ultimately degenerate. Viable cells can be reconstructed by PEG-induced fusion of karyoplasts to cytoplasts. Since reconstructed cells apparently do not segregate chromosomes, they may provide a clearer understanding of the interactions between the nucleus and the cytoplasm in the control of the expression of tumorigenicity. We have reconstructed cells using karyoplasts from the tumorigenic Y-1 cell line and cytoplasts from a nontumorigenic cell line, A-MT-BU-A1. In addition we have reconstructed cells containing Y-1 cytoplasts and A-MT-BU-A1 karyoplasts. The reconstructed cells porduced were assayed for tumorigenicity by their ability to grow in soft agar and in nude mice. The results of these experiments indicate that the reconstructed cells containing a tumorigenic nucleus and a nontumorigenic cytoplasm ultimately are tumorigenic and conversely the reconstructed cells containing a nontumorigenic nucleus and a tumorigenic cytoplasm are nontumorigenic. These experiments support the concept that with these cell lines the nucleus (karyoplast) is sufficient to control the phenotypic expression of tumorigenicity.     

Suppression of tumorigenicity in cybrids

We investigated cytoplasmic control of tumorigenicity in cybrids. Cytoplasts derived from nontumorigenic cells were fused to the highly tumorigenic 984 Cl 10–15 cell line derived from a murine teratoma. The resultant cybrids did not retain the tumorigenicity of the original cell line. In addition, the majority demonstrated the ability to differentiate into skeletal muscle. The results of these experiments indicate a heritable suppression of the tumorigenic phenotype by nontumorigenic cytoplasm. These findings are in contrast to our previous experiments in which we used a different experimental system and demonstrated a nuclear control of tumorigenicity in cybrids.     

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.     

Effects of reduced calcium on proliferation and cell viability in tumorigenic and nontumorigenic rat tracheal epithelial cell lines

We have compared the in vitro growth and viability of tumorigenic and nontumorigenic rat tracheal epithelial cell lines over a range of calcium concentrations from 0.003 to 0.85 mM. A greater dependence on calcium for proliferation was seen in the nontumorigenic line as compared to the tumorigenic line at both the colony formation level and in mass cultures. In the latter culture condition, a marked differential effect on cell survival was also demonstrated. These differences in calcium dependence were seen in media containing fetal bovine serum or low concentrations of newborn calf serum and in a serum-free medium developed for these cells. The effect was also independent of the method used for calcium removal i.e., either by chelex treatment or the inclusion of EGTA. Therefore, loss of calcium dependence may be associated with tumorigenicity in rat tracheal epithelial cells offering a selectable marker for neoplastic cells in carcinogen-exposed preneoplastic cell populations.     

Human Voltage-Gated Proton Channel Hv1: A New Potential Biomarker for Diagnosis and Prognosis of Colorectal Cancer

Solid tumors exist in a hypoxic microenvironment, and possess high-glycolytic metabolites. To avoid the acidosis, tumor cells must exhibit a dynamic cytosolic pH regulation mechanism(s). The voltage-gated proton channel Hv1 mediates NADPH oxidase function by compensating cellular loss of electrons with protons. Here, we showed for the first time, that Hv1 expression is increased in colorectal tumor tissues and cell lines, associated with poor prognosis. Immunohistochemistry showed that Hv1 is strongly expressed in adenocarcinomas but not or lowly expressed in normal colorectal or hyperplastic polyps. Hv1 expression in colorectal cancer is significantly associated with the tumor size, tumor classification, lymph node status, clinical stage and p53 status. High Hv1 expression is associated significantly with shorter overall and recurrence-free survival. Furthermore, real-time RT-PCR and immunocytochemistry showed that Hv1 is highly expressed in colorectal cancer cell lines, SW620, HT29, LS174T and Colo205, but not in SW480. Inhibitions of Hv1 expression and activity in the highly metastatic SW620 cells by small interfering RNA (siRNA) and Zn²⁺ respectively, markedly decrease the cell invasion and migration, restraint proton extrusion and the intracellular pH recovery. Our results suggest that Hv1 may be used as a potential biomarker for diagnosis and prognosis of colorectal carcinoma, and a potential target for anticancer drugs in colorectal cancer therapy.     

Discovery and validation of new Hv1 proton channel inhibitors with onco-therapeutic potential

The voltage-gated hydrogen channel Hv1 encoded in humans by the HVCN1 gene is a highly selective proton channel that allows large fluxes of protons across biological membranes. Hv1 form functional dimers of four transmembrane spanning proteins resembling the voltage sensing domain of potassium channels. Each subunit is highly selective for protons and is controlled by changes in the transmembrane voltage and pH gradient. Hv1 is most expressed in phagocytic cells where it sustains NADPH oxidase-dependent bactericidal function and was reported to facilitate antibody production by B cells and to promote the maturation and motility of spermatocytes. Hv1 contributes to neuroinflammation following brain damage and favors cancer progression possibly by extruding protons generated during aerobic glycolysis of cancer cells. Lack of specific Hv1 inhibitors has hampered translation of this knowledge to treat immune, fertility, or malignancy diseases. In this study, we show that the genetic deletion of Hv1 delays tumor development in a mouse model of granulocytic sarcoma and report the discovery and characterization of two novel bioavailable inhibitors of Hv1 channels that we validate by orthogonal assays and electrophysiological recordings.     

Tumor suppressor activity of profilin requires a functional actin binding site

Profilin 1 (PFN1) is a regulator of the microfilament system and is involved in various signaling pathways. It interacts with many cytoplasmic and nuclear ligands. The importance of PFN1 for human tissue differentiation has been demonstrated by the findings that human cancer cells, expressing conspicuously low PFN1 levels, adopt a nontumorigenic phenotype upon raising their PFN1 level. In the present study, we characterize the ligand binding site crucial for profilin's tumor suppressor activity. Starting with CAL51, a human breast cancer cell line highly tumorigenic in nude mice, we established stable clones that express PFN1 mutants differentially defective in ligand binding. Clones expressing PFN1 mutants with reduced binding to either poly-proline-stretch ligands or phosphatidyl-inositol-4,5-bisphosphate, but with a functional actin binding site, were normal in growth, adhesion, and anchorage dependence, with only a weak tendency to elicit tumors in nude mice, similar to controls expressing wild-type PFN1. In contrast, clones expressing a mutant with severely reduced capacity to bind actin still behaved like the parental CAL51 and were highly tumorigenic. We conclude that the actin binding site on profilin is instrumental for normal differentiation of human epithelia and the tumor suppressor function of PFN1.     

Proliferative responses of epithelial cells to 8-bromo-cyclic AMP and to a phorbol ester change during breast Pathogenesis

We have explored the relationship of changes in proliferative responses of human mammary epithelial cells to a phorbol ester (TPA) and to 8-Br-cAMP, which modulate the activities of protein kinases A and C (PKA and PKC), with breast tumour progression. Treatment with TPA had no effect on nontumorigenic cell lines established from human fibrocystic biopsies and apparently normal tissue around a tumour. In contrast, TPA strongly inhibited the proliferation of numerous human tumorigenic breast cell lines. Treatment with 8-Br-cAMP decreased the proliferation of all studied nontumorigenic and tumorigenic cell lines. We have also studied the effect of TPA and 8-Br-cAMP on growth of epithelial cells in short-term culture obtained from surgical human mammary biopsies with different states of breast disease. Both drugs enhanced growth of normal breast cells but had no significant effects on cells from biopsies with benign breast disease. In contrast, all examined cuitures from breast cancer biopsies were strongly inhhited by 8-Br-cAMP. Otherwise, TPA had an inhibitory effect only in the case of invasive ductal carcinoma of grade III. Malignant Ha-ras-transformation of nontumorigenic TPA-insensitive breast HBL-100 cells induced an inhibitory effect of TPA. In addition, a TPA-insensitive MCF7 clone was much less tumorigenic in athymic mice than the parental strain shown to be inhibited by TPA. These data suggest that the two intracellular transduction pathways change at different stages of breast pathogenesis. Alterations in the PKA pathway are early events and are probably important to cell immortalization but do not necessarily lead to malignant development. In contrast, changes in PKC pathway are rather later events associated with advanced malignant transformation. © 1994 Wiley-Liss, Inc.     

Functional evidence for a second tumor suppressor gene on human chromosome 17.

The development and progression of human tumors often involves inactivation of tumor suppressor gene function. Observations that specific chromosome deletions correlate with distinct groups of cancer suggest that some types of tumors may share common defective tumor suppressor genes. In support of this notion, our initial studies showed that four human carcinoma cell lines belong to the same complementation group for tumorigenic potential. In this investigation, we have extended these studies to six human soft tissue sarcoma cell lines. Our data showed that hybrid cells between a peripheral neuroepithelioma (PNET) cell line and normal human fibroblasts or HeLa cells were nontumorigenic. However, hybrid cells between the PNET cell line and five other soft tissue sarcoma cell lines remained highly tumorigenic, suggesting at least one common genetic defect in the control of tumorigenic potential in these cells. To determine the location of this common tumor suppressor gene, we examined biochemical and molecular polymorphic markers in matched pairs of tumorigenic and nontumorigenic hybrid cells between the PNET cell line and a normal human fibroblast. The data showed that loss of the fibroblast-derived chromosome 17 correlated with the conversion from nontumorigenic to tumorigenic cells. Transfer of two different chromosome 17s containing a mutant form of the p53 gene into the PNET cell line caused suppression of tumorigenic potential, implying the presence of a second tumor suppressor gene on chromosome 17.     

DNA methyltransferase levels in tumorigenic and nontumorigenic cells in culture

The levels of DNA methyltransferase in nuclei from 9 tumorigenic and 9 nontumorigenic cell lines were examined. In all but 2 cases, the extractable methyltransferase activity was 4-3000-fold higher in tumorigenic than in nontumorigenic cells. Tumorigenic and nontumorigenic cells from four species were grown in the presence of various concentrations (10(-8)-10(-6) M) of an inhibitor of the methylase enzyme, 5-aza-2'-deoxycytidine (5-aza-dCyd). The reduction of 5-methylcytosine content in newly replicated DNA in the presence of 5-aza-dCyd was used to determine the relative methylase activity in each cell line. In all 4 cases, tumorigenic cells required larger doses of drug to inhibit DNA methylation to the same extent as their nontumorigenic counterparts. The relative rates of incorporation of [3H]5-aza-dCyd were determined for each cell line, and tumorigenic cells were shown to incorporate equal or greater amounts of 5-aza-dCyd into DNA compared to nontumorigenic cells. These results showed that the differences in the inhibition of DNA methylation in response to 5-aza-dCyd were not due to differences in the ability of these cells to incorporate the drug. Thus, it was demonstrated by two independent methods that tumorigenic cells contained higher levels of methylating capacity than nontumorigenic cells. This overabundance of methyltransferase may alter DNA methylation patterns and affect phenotypic stability.     

Functional Reconstitution of Purified Human Hv1 H Channels

Voltage-dependent H⁺ (Hv) channels mediate proton conduction into and out of cells under the control of membrane voltage. Hv channels are unusual compared to voltage-dependent K⁺, Na⁺, and Ca²⁺ channels in that Hv channel genes encode a voltage sensor domain (VSD) without a pore domain. The H⁺ currents observed when Hv channels are expressed heterologously suggest that the VSD itself provides the pathway for proton conduction. In order to exclude the possibility that the Hv channel VSD assembles with an as yet unknown protein in the cell membrane as a requirement for H⁺ conduction, we have purified Hv channels to homogeneity and reconstituted them into synthetic lipid liposomes. The Hv channel VSD by itself supports H⁺ flux.     

Identification of a human chromosome 11 gene which is differentially regulated in tumorigenic and nontumorigenic somatic cell hybrids of HeLa cells.

The tumorigenicity of HeLa cells in nude mice can be suppressed by the addition of a normal human chromosome 11 in somatic cell hybrids. We have attempted to identify specific genes involved in this phenomenon by transfecting a complementary DNA expression library into a tumorigenic HeLa-fibroblast hybrid. A cell line designated F2 was isolated which displayed morphological features of the nontumorigenic hybrids, demonstrated reduced tumorigenicity in nude mice, and showed an 85% reduction in alkaline phosphatase, a consistent marker of the tumorigenic phenotype in these cells. F2 contained a single exogenous complementary DNA, which was recovered by polymerase chain reaction and designated HTS1 because of its potential association with "HeLa tumor suppression." Northern blot studies suggested differential regulation of the HTS1 gene dependent on the tumorigenicity of the cell. In nontumorigenic hybrids, RNA species of 2.8, 3.1, and 4.6 kilobases were identified. In two tumorigenic hybrid lines, the 2.8-kilobase species was markedly reduced or absent. Similarly, three nontumorigenic human keratinocyte lines expressed all three RNA species, whereas several tumorigenic cervical carcinoma cell lines lacked the 2.8-kilobase species. Chromosome localization studies mapped the HTS1 gene to chromosome 11p15, a region of chromosome 11 that is believed to contain a tumor suppressor gene. These findings indicate that HTS1 represents a novel chromosome 11 gene which may be a target of the tumor suppressor gene active in this system.     

Inhibition of growth by transforming growth factor-beta following fusion of two nonresponsive human carcinoma cell lines. Implication of the type II receptor in growth inhibitory responses.

Loss of growth regulation by transforming growth factor-beta (TGF-beta) may be an important step in carcinogenesis. We have used a cell fusion system to show that inhibition of growth by TGF-beta can be restored to carcinoma cell lines that are unresponsive to the inhibitory effects of TGF-beta. In a previous study, the EJ bladder carcinoma line was fused to the SW480 colon adenocarcinoma line and found to produce nontumorigenic hybrid cells along with one hybrid cell clone of low tumorigenicity. Here we show that the capacity of the nontumorigenic hybrid cells to respond to either TGF-beta 1 or TGF-beta 2 has been restored, while the parental or tumorigenic hybrid cells show little or no inhibition of growth following TGF-beta treatment. Cross-linking analyses with labeled TGF-beta 1 demonstrated much higher levels of the type II (85 kDa) receptor in the hybrid cells compared with the parental tumor lines. Both the parental and tumorigenic hybrid cell lines were capable of responding to TGF-beta as evidenced by increased levels of mRNA for fibronectin, type IV collagenase, and plasminogen activator inhibitor after treatment with TGF-beta 1. These results suggest that the type II receptor is necessary for mediating the effects of TGF-beta on inhibition of growth but not on gene activation of the hybrid cells.     

Correlation of the inability to sustain growth in defined serum-free medium with the suppression of tumorigenicity in Wilms' nephroblastoma.

We report the investigation of the growth properties of tumorigenic and reverted nontumorigenic Wilms' nephroblastoma cells when cultured in serum-free medium. Wilms' tumor, a pediatric nephroblastoma, has been associated with deletions encompassing the p13 band of chromosome 11 and an independent loss of heterozygosity at 11p15. Weissman et al. (Science 236:175-180, 1987) transferred a human der(11) chromosome into the G401.6TG.6 Wilms' tumor cell line via the microcell-mediated chromosome transfer technique. The resulting microcell hybrids were nontumorigenic when assayed in nude mice; however these cells retained all of the in vitro growth and morphological characteristics of the tumorigenic parental cells in 10% fetal calf serum (FCS). Segregation of the der(11) chromosome from the nontumorigenic microcell hybrid cells resulted in the reappearance of the tumorigenic phenotype in vivo. In vitro culture of these cell lines in serum-free medium supplemented with 0.1% bovine serum albumin (BSA) and 10 ng/ml Na2O3Se resulted in sustained growth of both the tumorigenic parent and the tumorigenic segregant while the nontumorigenic microcell hybrids were unable to divide. The separate addition of either 10 ng/ml of epidermal growth factor (EGF) or 5 micrograms/ml of insulin did not alter this effect. However, the addition of 5 micrograms/ml of transferrin stimulated the nontumorigenic microcell hybrid cells to grow at a rate comparable to the tumorigenic cells. In addition, conditioned serum-free medium from the tumorigenic parental or tumorigenic segregant cell lines was able to stimulate the growth of the nontumorigenic microcell hybrid cells, whereas the reciprocal experiment had no effect on the growth of the tumorigenic cells. These data suggest that the inability of the microcell hybrid cells to grow in serum-free conditions is correlated with their genetic nontumorigenic phenotype and that a specific growth factor, transferrin, can bypass or alter this negative growth regulatory pathway(s) in vitro.     

Inhibition of voltage-dependent Na+ current in cell-fusion hybrids containing activated c-Ha-ras

Summary The electrophysiological properties of EJ (human bladder carcinoma), GM2291 (human fetal lung fibroblast), and of three hybrid cell lines obtained from their cell fusion were investigated using the patch-clamp technique. GM2291 cells, which are nontumorigenic, express voltage-dependent Na⁺ channels. The pharmacology and gating properties of the Na⁺ channels in GM2291 cells are distinct from neuronal and cardiac Na⁺ channels. EJ cells, which are tumorigenic and contain activated c-Ha-ras, express inward rectifier K⁺ channels. The three cell-fusion hybrid lines, named 145 (nontumorigenic), 145L (non-tumorigenic but morphologically altered), and 147TR2 (fully tumorigenic segregant), have been previously shown to express levels of activated c-Ha-ras similar to those of the EJ parental line. Voltage-dependent Na⁺ channels were observed in none of the hybrid cell lines, while inward rectifier K⁺ channels were observed in each of the hybrid cell lines. The possibility that c-Ha-ras inhibits expression of a voltage-dependent Na⁺ channel is discussed.