This study illustrated that the cellular distribution of v-ATPase in human pancreatic cancer tissues may influence cancer cell activity since polarity is lost and expression was increased with advancing malignant features. In fact, striking differences in v-ATPase polarity and staining intensity distinguished early from advanced PanIN lesions. Invasive pancreatic cancers and metastatic lesions meanwhile demonstrated uniformly diffuse and intense v-ATPase staining. These findings indicate that elevated expression and loss of v-ATPase polarity may be key steps in modulating the tumor microenvironment, thereby providing a clinical correlate to previous in vitro
work in breast cancer cells that identified v-ATPase expression as a marker of cancer cell aggressiveness.11
A previous study in human pancreatic cancer specimens compared mRNA levels and immuno-labeling of the v-ATPase V0
c subunit in PDAC in relation to precursor lesions and benign cystic tumors.32
This study indicated that the mRNA levels of this subunit were increased in PDAC eight-fold over normal pancreas. Similar to the results reported here, the intensity of v-ATPase expression was highest in PDAC. The absence of positive staining in non-invasive cancers or in benign cystic neoplasms in this previous study is a notable difference from our results. The current study found that PanIN lesions demonstrated prominent v-ATPase labeling with a clear loss of polarity that coincided with increasing malignant features. Our current results demonstrate a unique pattern to v-ATPase (V1
E subunit) labeling within PDAC precursors, which suggests an early role for v-ATPase function in cancer cell homeostasis and invasive capacity as corroborated by previous literature.11, 19, 33
Future evaluation of v-ATPase expression in human cancer sections using standardized antibodies and techniques may be necessary to resolve these differences.
Other studies have demonstrated that the v-ATPase on plasma membranes contributes to acidification of the extracellular space which promotes invasive properties.11, 34
Targeted inhibition of the V0
c subunit led to diminished MMP-2 expression and reduced hepatocellular carcinoma growth in an animal model indicating that the therapeutic potential of inhibiting the v-ATPase may be due in part to reducing MMP-2 activity.34, 35
However, whether these findings are relevant to other types of cancers and other MMPs is unclear. We demonstrated that the v-ATPase is present on plasma membranes of Panc-1 cells; these cell lines have been described to have invasive potential in vivo
In Panc-1 cells, the v-ATPase co-localizes with cortactin, a component of the cellular invasion apparatus implicated in focal MMP-9 release.20, 21
Significantly, MMP-9 activity was reduced with v-ATPase blockade in three pancreatic cancer cell lines, but was least affected in BXPC3 cells which demonstrated little v-ATPase PM localization. Targeted inhibition of the V1
E subunit using shRNA constructs confirmed these findings in Panc-1 cells. Thus, specific pancreatic cancer cells display v-ATPase plasma membrane localization and MMP-9 activities that are v-ATPase dependent.
In contrast to MMP-9, concanamycin and bafilomycin inhibition of the v-ATPase increased the fully active MMP-2 isoform. A potential explanation for this difference may be the differential regulation and activation of MMP-2 and -9 precursors. MT1-MMP, a cell surface activator of MMP-2, is rapidly and constitutively down-regulated through a v-ATPase-dependent degradation process.31, 36
V-ATPase blockade results in increased levels of active MT1-MMP on the cell surface thereby amplifying MMP-2 activities, which is consistent with our findings.31, 37
These findings indicate that potential targeting of the v-ATPase in cancer studies may have indirect (and unintended) effects on regulators of MMP activation that could enhance, rather than block, specific protease activities.
Since MMP-2 activation with concanamycin treatments reflects the ability of intact intracellular degradation pathways that are v-ATPase dependent, the current studies using chemical v-ATPase inhibitors do not allow us to discern the relative contributions of extracellular versus intracellular proton flux. Future experiments targeting the specific subunits associated with plasma membrane localization may help to provide additional evidence supporting a role for v-ATPase-mediated extracellular acidification.
Although we have shown that the v-ATPase can modulate MMP activities in cancer cells, this transporter can affect other cellular responses that may be relevant to cancer biology. For instance, v-ATPases may mediate resistance to chemotherapeutic agents. Tumor cells, when exposed to chemotherapeutic drugs, demonstrate transcriptional promoter activity that leads to the induction of specific v-ATPase levels.38, 39
This induction precludes cancer cell apoptosis in response to chemotherapy demonstrating that v-ATPase expression may be a protective mechanism against chemically-induced apoptosis. Combined application of chemotherapy and a v-ATPase inhibitor restored the ability of these agents to cause cancer cell apoptosis.39
Thus, in addition to effects on MMP activities, targeting v-ATPases could also enhance the sensitivity of cancer cells to chemotherapeutics.
Accumulating evidence also points to the importance of cell surface v-ATPase function in non-malignant processes. In renal tubular epithelium, the v-ATPase is vital for urine acidification. Osteoclasts are enriched with specific v-ATPase isoforms at the ruffled border, which enables an optimal pH environment for proteases to degrade matrix. Endothelial cells adjacent to an induced wound display inducible and prominent plasma membrane v-ATPase localization and activity.10
Endothelial cells obtained from diabetic rat models, however, display significantly reduced cell surface v-ATPase activity and show a functional impairment in migratory behavior.40
These findings indicate that physiological and wound-repair processes likely require plasma membrane v-ATPase activity, possibly due to the effects on MMPs.
In summary, v-ATPase staining of human pancreatic malignancy, ranging from PanIN lesions to PDAC, demonstrated a marked loss of polarity and increased intensity with increasing tumor invasiveness. These changes were seen in PanIN lesions suggesting a role for the v-ATPase in early stages of malignant transformation. Inhibiting v-ATPase function decreased MMP-9 activities, but increased MMP-2 activation in vitro. These results indicate that the v-ATPase plays a complex role in regulating MMPs and the pancreatic cancer phenotype.