In this post I will discuss the role of human Hv1 channels in promoting the pathology and invasiveness of breast cancers. It is well known that cancer consists of normal cells gone awry. As cells age, they can accumulate mutations in their genomes and sometimes these mutations lead to unregulated growth and expansion. The unregulated, cancerous cells require a great deal of energy to produce their expansive growth and therefore many cancers are characterized by increased levels of metabolism, specifically of glycolysis. Because cancerous cells become dense tumors, the microenvironment inhabited by the cells can be very low in oxygen (hypoxic). High metabolism plus hypoxia means that the cells quickly start to build up high levels of protons (a byproduct of anaerobic glycolysis) in their cytoplasm (Gatenby & Gillies, 2007). Without a mechanism for dumping protons out of the cytoplasm, cancer cells would not be able to efficiently proliferate. When a cancer cell is able to efficiently proliferate it can spread from its primary site to other locations throughout the body, a process known as metastasis (for more information on this process check out metastasis on wikipedia). People are only beginning to tease apart the processes employed by different cancers to regulate their internal pH and become highly metastatic. One mechanism that is becoming apparent in certain types of breast cancer is the over-expression of human Hv1. Here I will discuss two recent papers by Wang et al. that try to dissect the role of human Hv1 in breast cancer metastasis and clinicopathology (Wang et al., 2011; Wang et al. 2012).
Human Hv1 is overexpressed in highly metastatic breast cancer cell lines
The discovery by Wang et al. of specific expression of human Hv1 in highly metastatic breast cancer cells—both in human tissue samples obtained from breast cancer surgery and in breast cancer derived cell lines—has led to the hypothesis that these channels are responsible for the regulation of cytoplasmic pH in these cells (Wang et al., 2011). What is most interesting from this first study is the specificity of the expression pattern, in which high levels of human Hv1 expression is only seen in the highly metastatic breast cancers and cell lines (Wang et al., 2011). This suggests that the presence of the channel in the membranes of cancer cells may help them be more invasive.
To further investigate this possibility, Wang et al. generated a series of short interfering RNAs (siRNAs) that they used to reduced the expression of the channel in the cancer cell lines (Wang et al., 2011). When a highly metastatic cell line was treated with one of these siRNAs, it showed a significant decrease in migration and invasion, two hallmarks of highly metastatic cells. They also showed that the internal pH of the cells decreased, indicating an inability of the cells to properly remove excess protons from their cytoplasm (Wang et al., 2011).
It has been demonstrated that the activity of the gelatinase proteases in the extracellular matrix (ECM) both facilitates the metastasis of cancer cells and is pH-regulated (Giannelli & Antonaci, 2002; Gioia et al., 2010). It is thought that these enzymes remodel the ECM, which in turn promotes growth and metastasis of the tumor. Wang et al. also show, using their human Hv1 siRNAs, that the activity and expression of these enzymes are reduced when the expression of human Hv1 is knocked down.
Although this initial study suggests that the presence of human Hv1 is correlated to metastasis—and even suggests a mechanism through which disrupting pH regulation reduces migration and invasion; and disrupts the activity of gelatinases—Wang et al. are missing some very important control experiments. What they are missing is a “rescue” experiment, i.e., they do not demonstrate that, by reintroducing an siRNA-resistant human Hv1 into the siRNA-treated cells, the migration, invasion and gelatinase activity levels return to control levels. In addition, they do not show whether, by introducing human Hv1 to a poorly metastatic breast cancer cell line, they increase these cells’ ability to migrate and invade surrounding tissues. Without these controls we can’t be sure whether the effect shown in this study is directly from the activity of the Hv channels or from some secondary, off-target effect of the siRNA. Without these rescue experiments, Wang’s study remains inconclusive.
Overexpression of Hv1 correlates with breast cancer clinicopathology
In a follow up study, Wang et al. examined 105 different breast cancer tissues taken from patients who underwent routine curative surgery (Wang et al. 2012). Each tissue sample was paired with a sample of normal breast tissue from the same patient and both samples were examined for overexpression of human Hv1 channels in a blinded manner (i.e. the clinical pathologist examining the sample did not know any of the clinical information prior to examination) (Wang et al. 2012). This large data set contained several different types of breast cancers and showed a wide range of human Hv1 expression. What was determined was that high levels of human Hv1 correlated with poor prognosis. In other words, the more Hv channels expressed in the tumor cells, the more deadly the cancer (Wang et al. 2012).
Some of the other data in this paper is very similar to that discussed above for their 2011 siRNA study (Wang et al., 2011). However, here they probe the physiological role that Hv channels may be playing in the cells in more detail. Using pH sensitive fluorescent dyes, they show, that when human Hv1 is knocked down the ability of the cancer cells to regulate their cytoplasmic pH is decreased. In addition, the cells’ ability to acidify the extracellular medium also decreases (Wang et al., 2011). Again, they show that cells treated with Hv1 siRNAs have reduced gelatinase activity (Wang et al., 2011), and again the control rescue experiments were not performed.
In the final experiment of the study, Wang et al. perform xenografts of the cultured tumor cells onto immunodeficient “nude” laboratory mice (check them out on wikipedia). They show that the tumors that grow on the mice are smaller if the cells are pre-treated with the human Hv1 siRNA. They also show that the tumors derived from the cells treated with siRNA continue to have low Hv channel expression levels (Wang et al., 2011).
These studies clearly show that human Hv1 is overexpressed in certain breast cancers and that the level of Hv channel expression is correlated with prognosis. The more Hv channels a tumor has, the more likely that tumor is to be highly metastatic and deadly. However, a causal link between Hv channels and breast cancer severity is not established. The experiments using siRNA knockdown of human Hv1 are inconclusive. There are well-established standard for these sorts of RNA interference (RNAi) knockdown experiments and you must include proper controls to prevent the occurrence of false positives (Echeverri et al., 2006). Wang et al. fail to perform these controls and hence do not conclusively show that it is in fact Hv channels that are responsible for the increase in cancer severity.
Off-target effects of siRNAs are more common than people are generally aware of and have led to the re-evaluation of high-profile papers several times (Hubner et al., 2010; Westhorpe et al., 2010). Regardless of this, every proper experiment needs its proper control. In siRNA studies, using a “scrambled” or irrelevant siRNA (e.g. GAPDH, lamin B, GFP) is not enough: rescue experiments should be the minimal standard.
It is clear that in order to be highly proliferative cancer cells need to develop mechanisms to maintain their cytoplasmic pH. It is reasonable to assume that overexpression of Hv channels would help provide cells with such a mechanism. This reasonableness and the correlation seen between Hv1 overexpression and clinicopathology certainly make an intriguing argument for the direct role of the channel in promoting metastasis. But as scientists we have to be skeptical. Before spending millions of dollars to develop specific Hv blockers for cancer therapy, I would certainly like to see the control experiments. However, if the control experiments are performed and demonstrate that Hv current directly contributes to metastasis, then drugs that target and block Hv channels could be very promising in the future treatment of breast cancer.
More research still needs to be done.
Works Cited and Further Reading
Echeverri, C. J., Beachy, P. A., Baum, B., Boutros, M., Buchholz, F., Chanda, S. K., Downward, J., et al. (2006). Minimizing the risk of reporting false positives in large-scale RNAi screens. Nature Methods, 3(10), 777–779. doi:10.1038/nmeth1006-777
Gatenby, R. A., & Gillies, R. J. (2007). Glycolysis in cancer: A potential target for therapy. The International Journal of Biochemistry & Cell Biology, 39(7-8), 1358–1366. doi:10.1016/j.biocel.2007.03.021
Giannelli, G., & Antonaci, S. (2002). Gelatinases and their inhibitors in tumor metastasis: from biological research to medical applications. Histology and histopathology, 17(1), 339–345.
Gioia, M., Fasciglione, G. F., Monaco, S., Iundusi, R., Sbardella, D., Marini, S., Tarantino, U., et al. (2010). pH dependence of the enzymatic processing of collagen I by MMP-1 (fibroblast collagenase), MMP-2 (gelatinase A), and MMP-14 ectodomain. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 15(8), 1219–1232. doi:10.1007/s00775-010-0680-8
Hübner, N. C., Wang, L. H.-C., Kaulich, M., Descombes, P., Poser, I., & Nigg, E. A. (2010). Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs. Chromosoma, 119(2), 149–165. doi:10.1007/s00412-009-0244-2
Wang, Y., Li, S. J., Pan, J., Che, Y., Yin, J., & Zhao, Q. (2011). Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis. Biochemical and biophysical research communications, 412(2), 353–359. doi:10.1016/j.bbrc.2011.07.102
Wang, Y., Li, S. J., Wu, X., Che, Y., & Li, Q. (2012). Clinicopathological and biological significance of human voltage-gated proton channel Hv1 over-expression in breast cancer. Journal of Biological Chemistry. doi:10.1074/jbc.M112.345280
Westhorpe, F. G., Diez, M. A., Gurden, M. D. J., Tighe, A., & Taylor, S. S. (2010). Re-evaluating the role of Tao1 in the spindle checkpoint. Chromosoma, 119(4), 371–379. doi:10.1007/s00412-010-0261-1