This study demonstrates that, despite close similarities between the HIF-α isoforms, differential activation of HIF-1α or HIF-2α pathways in VHL-defective RCC cells has nonequivalent or even opposing effects on gene expression and experimental tumor growth. Our investigations differ somewhat from previous studies of the role of HIF in RCC tumor growth, in which two different groups used either mutated HIF-1α (27
) or mutated HIF-2α molecules (18
), which escape VHL recognition, to test for opposition of the tumor-suppressive effect of reintroducing wild-type VHL into 786-O cells. We used wild-type HIF-α isoforms in assays for effects on tumor growth and directly compared the effects of augmenting HIF-1α or HIF-2α on the growth of 786-O cells as subcutaneous tumors in nude mice. Our data indicate that the apparent discrepancy between the two previous studies arises from real differences between the actions of HIF-1α and HIF-2α, with HIF-2α having positive effects on tumor growth and HIF-1α having negative effects on tumor growth in this setting. Since we also demonstrate suppressive interaction between HIF-α isoforms, we cannot as yet distinguish whether suppression of tumor growth by HIF-1α arises from direct effects, indirect effects due to downregulation of HIF-2α, or a combination of both possibilities. Interestingly, in contrast to effects on tumor growth, no effects were observed on monolayer growth under standard tissue culture conditions, as has been observed in assays of VHL tumor suppressor function based on re-introduction of wild-type VHL into VHL-defective RCC cells (12
Consistent with differential effects on tumor growth were differential effects on the expression of specific genes with putative pro- and antitumorigenic effects. In particular, HIF-1α positively regulated BNip3 but had no effect on cyclin D1, TGF-α, and VEGF, whereas HIF-2α negatively regulated BNip3 and positively regulated cyclin D1, TGF-α, and VEGF. BNip3 is a member of the Bcl-2 family of apoptosis-regulating proteins and activates caspase-independent necrosis-like cell death by opening the mitochondrial permeability transition pore (43
). In most cells, the protein is strongly induced by hypoxia, and an involvement in hypoxic tumor necrosis has been postulated (3
). Cyclin D1 is one of the main G1
-phase cyclins. Its expression is associated with G1
-to-S transition in the cell cycle and upregulated in many types of cancer by a variety of mechanisms (38
). TGF-α, on the other hand, is a potent renal cell mitogen that activates the epidermal growth factor receptor pathway and has been proposed to initiate an autocrine loop with this receptor when VHL is inactivated in renal cells (6
). CAIX and GLUT-1 are both upregulated in many forms of cancer, but their contribution to tumor growth is less clear. GLUT-1 was found to be a specific HIF-2α target in VHL-defective RCC but not in other cells, whereas CAIX was found to be a specific HIF-1α target in both RCC and non-RCC cells, as has been reported for genes encoding a range of glycolytic enzymes (11
). Interestingly, though upregulation of CAIX is commonly observed in RCC (25
), reduced staining has been correlated with poorer prognosis in RCC in a recent clinical series (4
Based on known functions, it seems likely that the HIF-α isoform transcriptional selectivity manifest by one or more of the genes analyzed could contribute to differential effects of the HIF-α isoforms on VHL-associated RCC growth. However, the exact contribution of individual genes was not defined. Given the complexity of the HIF transcriptional cascade and the large number of direct and indirect HIF targets identified in gene expression arrays (11
), such an analysis remains a considerable task. Nor can it be deduced that other potentially protumorigenic and antitumorigenic HIF target genes will fall into the above pattern, particularly as in non-RCC settings the balance of evidence favors a positive action of HIF-1α on tumor growth (37
). Presumably, the set of HIF-1α and HIF-2α targets in part reflects differences in the physiological role of these proteins in the response to hypoxia that are as yet unclear. Interestingly, HIF-1α has recently been demonstrated to antagonize Myc and induce cell cycle arrest by complexing Myc in a transcriptionally inactive form (20
). In future studies it will be interesting to determine if this property is shared by HIF-2α and whether it contributes to effects on RCC tumor growth.
Though HIF-α transcriptional selectivity was observed in all cells, there were important differences between what were essentially two distinct patterns, that observed in VHL-defective RCC cells and that observed in all other cells, including the VHL-competent RCC line Caki-1. Differences in transcriptional targeting were of two types. First, some genes such as cyclin D1 appeared to be transcriptional targets of HIF-2α only in VHL-defective cells. TGF-α may also fall into this category but has been less extensively studied in VHL-competent cells. Second, some genes, such as those for GLUT-1 and VEGF, appeared to be specific HIF-2α targets in VHL-defective RCC cells whereas in VHL-competent Caki-1 and non-RCC cells they appear potentially responsive to both HIF-α isoforms. For instance, for GLUT-1, HIF-α suppression by siRNA in Caki-1 and non-RCC cells indicates clear dependence on HIF-1α, whereas overexpression studies show that enhanced expression of both isoforms, but particularly HIF-2α, can clearly drive GLUT-1 expression. These findings correlate well with published work with other non-RCC cells that has shown that GLUT-1 is induced by overexpression of both HIF-α isoforms in VHL wild-type HEK 293 cells (11
) but predominantly dependent on endogenous HIF-1α, as revealed by genetic inactivation studies (39
). In contrast, in VHL-defective RCC cells, neither HIF-1α siRNA nor HIF-1α overexpression had any effect on GLUT-1, even in cells retaining substantial levels of HIF-1α that was transcriptionally active on other target genes. Though the mechanistic basis of this difference is unclear, the simplest explanation is that a specific transcriptional connection between HIF-1α and these genes is missing in VHL-defective RCC.
It is difficult to distinguish whether these unusual properties of the HIF system are newly acquired during RCC development as further events following VHL inactivation or whether they reflect unusual intrinsic properties of the cells giving rise to VHL-associated RCC. The clear differences from the related renal cell line Caki-1 favor the occurrence of additional events, though concordance across a number of RCC lines suggests that such events must occur at high probability following VHL inactivation.
Also of interest in understanding the unusual properties of the HIF system in VHL-defective RCC cells is the demonstration of suppressive interactions between HIF-α isoforms. Though a natural antisense to the 3′ untranslated region of HIF-1α has been described (aHIF) (41
) that may suppress HIF-1α mRNA in RCC cells, the suppressive interaction we observed was not manifest on the mRNA level, indicating that it represents a different process. Our results are consistent with an action on HIF-α protein translation, though we have not yet defined the mechanism. Whether such a process extends to other components of hypoxia pathways such as the translation of HIF target genes is unclear, though in general we found that changes in expression of HIF target gene transcripts and protein products were concordant. Whatever the mechanism, our analysis suggests that the interaction we describe is not a distinct property of the HIF system in VHL-defective cells; rather, it is uncovered as a direct consequence of VHL inactivation blocking the HIF degradation pathway, which is saturable, leading to competitive interactions between HIF-1α and HIF-2α in the opposite direction of that seen in VHL-competent cells. Nevertheless, this phenomenon could well contribute to the unusual dominant expression of HIF-2α over HIF-1α expression observed in VHL-defective RCC, since upregulation of HIF-2α would now be anticipated to downregulate HIF-1α and vice versa. Such a scenario begs the following question: is upregulation of HIF-2α or downregulation of HIF-1α the primary event in driving the observed bias? Currently, we cannot answer this question, though it is of interest that a recent report has described a shortened HIF-1α transcript in 786-O cells (44
) and in previous work we have observed a shortened HIF-1α protein species in A498 cells (M. E. Cockman, Ph.D. thesis, Oxon, 2003), suggesting that at least in some cases inactivation of HIF-1α may be a discrete event.
Overall, our data indicate that HIF-1α and HIF-2α have functionally distinct roles in the biology of VHL-defective RCC, with HIF-2α promoting and HIF-1α retarding tumor growth, and that the HIF system behaves unusually in this setting in a number of ways. There is growing interest in the development of HIF inhibitors as anticancer agents (24
). Our findings clearly have relevance to targeting the HIF system in cancer therapy and emphasize the importance of considering isoform-specific effects.