**10. V-ATPase and cancer**

As mentioned earlier, V-ATPase pump activity and function has been associated with various diseases. However, one such non genetic disease that these pumps have been linked to is cancer [106]. Normal V-ATPase pump functioning is crucial for various signaling pathways in the cell. In fact, many of the pathways that lose their homeostatic control during cancer require V-ATPase pumps for proper functioning, such as the Notch signaling pathway and the Wnt/β-catenin signaling pathway [106–108]. Moreover, proper pump activity is required for the activation of the mechanistic target of rapamycin complex, or mTORC1, which is a common pathway implicated in cancer [109–113]. However, studies have shown more direct roles of V-ATPase pumps in cancer. For example, when V-ATPase pumps are inhibited in human tumor cells, results showed programmed cell death is induced [114–119]. These findings indicate that cancer cells rely on V-ATPase activity much more than noncancerous cells do in order to remain viable [115, 116, 119]. This is because cancerous cells have been shown to generate increased levels of acidity [120]. Since V-ATPase pumps play the critical role of removing acids out of the cell and help to preserve a neutral pH in the cytosol, cancer cells heavily rely upon V-ATPases to promote the alkalinization of their cytosol and to pump protons into the extracellular space [1, 2, 121]. It has been hypothesized that expression of these pumps may upregulate or may be specifically localized to the cellular membrane by expressing certain isoforms that target the V-ATPases to the plasma membrane [64, 65, 122, 123]. In this way, tumors are thus able to dodge programmed cell death and can proliferate [106]. In fact, V-ATPase pumps have been found in the plasma membrane of numerous invasive cancer cell lines while not in non invasive ones [124]. For example, an invasive line of breast tumor cells revealed higher levels of the V-ATPase pump on the cell surface compared to noninvasive breast cancer cells [124]. As explained above, these pumps aid in the maintenance of cytosolic pH. The impact of V-ATPases has also been observed in various other invasive cancerous cell lines, including liver cells, esophageal cells, ovarian cells, lung cells, prostate cells and pancreas cells amongst others [106, 110, 111, 125–129]. Research has shown that inhibiting V-ATPase pumps disrupts the pH balance and causes a more acidic pH in the cell which leads to higher levels of apoptosis [130].

Furthermore, V-ATPase pumps are not only important for regulating the pH of the cytosol, but for regulating the pH of other organelles as well [106]. Implications of protein production and shortages in nutrients can lead to ER stress in cancer cells. As a result, lysosomes are activated by the Bax inhibtor-1 in response to the

ER stress in order to raise protein turnover [131]. Studies have shown that blocking ATPase pump activity barred this from occurring and ultimately led to cell death [131]. Moreover, V-ATPases in the cell membrane have also been proposed to play a crucial role in the migration and invasion of cancer cells. Studies have shown that when V-ATPase pumps were inhibited in invasive breast cancer cells, the migration and invasiveness of these cells was diminished [1, 2, 124]. This was further proven in other cancer lines as well, such as cancerous pancreas cells [125]. Thus, the relationship between V-ATPases and cancer, and subsequently its link to apoptosis, has been an active area of research. However, the role of V-ATPases in yeast and its connection to apoptosis has not been as well characterized compared to that of human cell lines. This review will now look at a possible mechanism by which V-ATPase activity can be linked to apoptosis via its regulation by key players of the lipid biosynthetic pathway.

### **11. V-ATPase genes and the lipid biosynthetic pathway**

As explained earlier, inositol is one of the key phospholipid precursors whose presence is essential in regulating phospholipid metabolism [69–72]. When present in the growth medium, inositol production is repressed. If inositol is absent in the growth medium, then the genes involved in its production are upregulated. Research has shown that cells that lack one or more of the vacuolar membrane ATPase genes exhibited defects in growth when cultured in media without inositol [132, 133]. However, these defects were able to be reversed if *OPI1*, the gene that encodes the Opi1p repressor, was deleted [133]. Additionally, cells that lack one or more V-ATPase genes have higher buildups of oxidant molecules which may indicate their relationship to protecting the cell and their role in preventing cell death.

Recent studies have shown that one of the key vacuolar membrane ATPase genes, *VMA3*, plays a significant role in regulating the synthesis of phospholipids. Growth experiments that were performed with cell lines lacking the *VMA3* gene exhibited a growth defect that had cells growing much slower when cultured without inositol in their media compared to wildtype cells [134]. *VMA3*'s role in *de novo* phospholipid production was further clarified when mRNA studies showed *HXK2*, another important gene in the phospholipid biosynthetic pathway required for inositol production, had significantly lower mRNA levels in the cells lacking *VMA3* compared to wildtype. Thus, *VMA3* was revealed to impact the phospholipid biosynthetic pathway by specifically regulating the transcription of the *HXK2* gene [134]. This finding was particularly interesting since other studies have shown that cells lacking *HXK2* exhibit a growth sensitivity to acetic acid [135], which is a growth condition that has long been used to screen for apoptosis. Earlier studies have hypothesized that *HXK2* plays a role in shielding cells from programed cell death since cells that have had the *HXK2* gene deleted had an accrual of activated Ras by the mitochondria [135]. Thus, further experimentation from this study showed that *vma3∆* cells grew significantly slower in the presence of acetic acid compared to wildtype and were even more sensitive to this apoptotic inducing agent when grown without inositol present. Taken together, these findings indicate that the deletion of *VMA3* leads to decreased transcription of the *HXK2* gene, which ultimately leads to cells being more sensitive to acetic acid. This therefore demonstrates that *VMA3* plays an important regulatory role in apoptosis [134].

*HXK2* plays an important role in glucose metabolism and encodes the hexokinase-2 enzyme that catalyzes the reaction which converts glucose into glucose-6-phospate. Thus, *HXK2* is regulated by the presence of glucose. Studies have shown that *HXK2* is regulated by two important transcription factors, Rgt1 and

*The Interplay of Key Phospholipid Biosynthetic Enzymes and the Yeast V-ATPase Pump… DOI: http://dx.doi.org/10.5772/intechopen.97886*

Med8, that cause the deregulation of *HXK2* when glucose is not present [136]. The findings by Konarzweska *et al*. have now also shown that *HXK2* is also regulated by Vma3p. Thus, there is a clear relationship between V-ATPases and the phospholipid biosynthetic pathway since Vma3p has been shown to upregulate the *HXK2* gene. Furthermore, it has been shown that by regulating the *HXK2* gene, *VMA3* has an important protective role when it comes to acetic acid induced apoptosis. This is therefore an important link between how V-ATPases relate to the lipid biosynthetic pathway.
