The major findings in this report are: a) HLRCC tumors over express LDH-A; b) LDH-A inhibition results in increased apoptosis via ROS production in an A549 surrogate FH knockdown cell line; and c) inhibition of fermentative glycolysis in this cell line results in significant reduction in growth in a xenograft mouse model.
The common feature of tumor cells is their reliance upon fermentative glycolysis, a phenomenon coined as the Warburg effect (1
). Although the mechanism that may control this metabolic shift is not clearly defined, many lines of investigations suggest that HIF expression may be central to Warburg’s effect both by induction of fermentative glycolysis and by diminishing the activity of PDH (7
). FH inactivation should mimic this state, as it results in increased expression of HIF1 α and upregulaiton of HIF-dependent glycolysis (13
). This pseudohypoxic drive is similar to that described in VHL deficient cells as a result of the HIF accumulation in the absence of the appropriate ubiquitination process that targets HIF. The switch to the fermentative glycolysis should therefore be a common feature in tumors with VHL, FH, and SDH (succinate dehydrogenase) deficiency, resulting from HIF1 α stabilization, and we would expect to see increased LDH-A expression in these tumors. Indeed, we have shown this to be the case in two of these tumor types, namely VHL and FH deficient tumors.
The functional consequences of inhibiting LDH-A have been explored both in vitro and in vivo. In the background of FH deficiency, LDH-A knockdown cells proliferate slower, undergo apoptosis and are less invasive. The underlying basis for these effects remains to be fully defined. FH/LDH-A deficient cells did show a ~50–60% reduction in intracellular ATP. We were surprised that the levels were not more profoundly depressed, since both fermentative glycolysis, and TCA cycle were expected to be compromised in FH/LDH-A cells. It is possible that intracellular ATP levels are being sustained either by leakiness in our surrogate system, by LDH-B overactivity or expression compensating for LDH-A inhibition, or by diminished ATP consumption. We are currently exploring whether inhibition of both isoforms of LDH may result in significant ATP depletion. This might be technically challenging, as it will involve a triple knockdown to create such a cell line.
A correlation between apoptosis, ROS, and mitochondrial respiration had been reported in several cancers (36
). Studies have suggested that HIF1 α dependent mitochondrial repression may provide survival benefit by decreasing the risk associated with apoptotic cell death (37
). Moreover, reduced ROS levels and decreased apoptosis results from forced expression of HIF1 α in oral squamous cell carcinoma (38
). We therefore asked if increased ROS production was mediating apoptosis. This appears to be the case, at least partially, since NAC was able to decrease PARP cleavage. We also found that OXPHOS was enhanced in FH/LDH deficient cells – as evidenced by increased oxygen consumption. One effect of this might be to increase electron flow in OXPHOS and thereby account for increased ROS production. However, in preliminary observations, we did not find increased superoxide formation as measured by the Mitosox reagent (data not shown), so the source of increased ROS production remains to be defined. It is also possible that various anti-oxidant systems in these cells have also been affected accounting for increased ROS.
Another effect of the increased rate of oxygen consumption observed in FH/LDH-A deficient cells could be to maintain the NADH/NAD+
ratio. Since LDH-A catalyzes regeneration of NAD+
from NADH, we expected to observe an increased NADH/NAD+
ratio in FH/LDH deficient cells. The competitive phenomenon between mitochondrial NADH/NAD+
transport system and LDH-A for NADH consumption is driven by LDH-A as a result of its fast enzymatic kinetic properties and is thought to be a potential factor for decreased mitochondrial respiration (39
). Our failure to note an increase in the NADH/NAD+
ratio may be partly due to a decrease in the LDH-A driven competitive reaction that drives more NADH into mitochondria.
Since hypoxia regulates transcription factors HIF1 and HIF2, and in general it is difficult to design inhibitors targeting transcription factors, we have investigated whether targeting downstream targets of HIF1 and HIF2 may be therapeutically advantageous. Indeed, VEGF, a HIF1 α induced gene product has been successfully targeted for renal cell carcinoma therapy and our in-vitro
data showing enhanced VEGF expression in these cells supports the use of VEGF inhibitors in HLRCC. Our in vitro
and in vivo
data supports the notion that targeting LDH-A, another HIF1 α target may be a viable strategy, for treating this disease as well. Moreover, high lactate levels are associated with poor prognosis in advanced renal cell carcinoma (40
) and in head and-neck cancer (41
). It is likely that inhibition of LDH-A by increasing the extracellular pH (42
) may reduce the metastatic ability of these advanced cancer cells as extrapolated from our in vitro
invasion assay results. Also, HLRCC patients may benefit from combined anti-angiogenic and LDH-A inhibitor therapy, as might patients with any tumors that have upregulated HIF.
Our findings add to a growing literature that suggest that metabolism plays a key role in tumorigenesis and is linked either directly or indirectly to hallmarks of cancer that are involved in initiation and proliferation of tumors. In conclusion, our data support the hypothesis that inhibition of fermentative glycolysis might serve as a therapeutic strategy for HLRCC. Therefore, the development of inhibitors of LDH-A makes eminent sense for the treatment of HLRCC and a potentially large group of cancers in which the Warburg effect is operational, perhaps as many as 60–80% of tumor types.