Under hypoxic conditions the activation of HIF transcription factors enables cells to alter their metabolism and avoid stress-induced apoptosis. Aberrant HIF activity in the inherited renal cancers BHD, VHL, HLRCC and TSC, is linked to the expression of growth and pro-angiogenic factors that are important in tumour growth. A new report from Doonachar et al., (2015) focuses on the differential effects of the HIF-1? and HIF-2? isoforms on stress-induced apoptosis in two VHL-deficient renal cell carcinoma (RCC) cell lines.

Under normoxic conditions VHL protein forms part of the E3 ligase complex that targets HIF for degradation thereby limiting the production of HIF-target genes; HIF-1? primarily upregulates glycolytic genes and HIF-2? upregulates angiogenic and growth factors. Homozygous loss-of-function mutations in VHL, both inherited and sporadic, in renal cells results in an increase in either HIF-1? and HIF-2? activity or only HIF-2? activity, and subsequent tumour growth. HIF-2? is therefore seen as a major driver of renal tumourigenesis following the loss of a tumour suppressor such as VHL (Kondo et al., 2003).

VHL has also been shown to help protect renal cells from apoptosis following a range of stresses (Schoenfeld et al., 2000). Doonachar et al., aimed to determine any differential effects of HIF? isoform on apoptosis in VHL-defective cells following cellular stress.

In the VHL-deficient 786-O cell line the transgenic expression of mutant VHL, compared to transgenic wild type VHL, induced expression of HIF-2? (the only isoform this line expresses) and apoptosis following UV exposure. Inhibiting HIF-2? translation with shRNAs, confirmed through reduced target gene GLUT1 expression, marginally reduced apoptosis suggesting a partial protection from UV-induced apoptosis. The loss of HIF-2? in this cell line did alter the rate of cell death induced by glucose starvation or serum withdrawal. Under all stress conditions the expression of functional VHL drastically reduced 786-O cell death confirming it as anti-apoptosis factor.

In a second kidney tumour cell line, RCC10, that expresses both HIF? isoforms, the presence of only mutated VHL resulted in increased expression of HIF-1? and HIF-2? and a greater rate of apoptosis following UV exposure. Inhibiting transcription of either HIF-1? or HIF-2? via shRNA reduced respective protein levels but did not impact on GLUT1 expression suggesting transcriptional redundancy. shRNA-inhibition of HIF-1? increased cell death following UV exposure and glucose starvation suggesting that HIF-1? has an anti-apoptotic role. In contrast shRNA-inhibition of HIF-2? reduced cell death following UV exposure and serum withdrawal, indicative of a pro-apoptotic role. However, as there was also a, potentially compensatory, increase in HIF-1? expression following HIF-2? shRNA-inhibition, it could also be attributed to an increase in HIF-1? anti-apoptotic activity. There was no comparative increase in HIF-2? expression detected following shRNA-inhibition of HIF-1?.

Doonachar et al., conclude that HIF? isoforms seem to have different roles following cellular stress; HIF-1? is generally anti-apoptotic and HIF-2? pro-apoptotic. Further work is needed to determine if differences in isoform function are also relevant in tumour formation and if they could be exploited in treatments.

In contrast to VHL studies, Flcn-null cell lines show no increase in HIF-1? or HIF-2? expression despite a marked increase in activity determined by increased expression of target genes (Preston et al., 2010, Yan et al., 2014). The increase in HIF activity reported in BHD RCCs is thought to be mediated through AMPK and PGC1? activity. Yan et al., propose that FLCN loss increases AMPK and PCG1? activity leading to increased mitochondrial biogenesis, increased ROS production and subsequent activation of HIF signalling. Mutations in FLCN have also been linked to aberrant mTOR activity (Baba et al., 2006), an upstream activator of HIF signalling in tumour cells (Hudson et al., 2002). It is possible that these mechanisms, alongside increased HIF protein stability resulting from reduced VHL-dependent ubiquitination and degradation, drive enhanced HIF signalling in VHL-RCCs.

Increased HIF activity has also been reported in BHD pulmonary cyst epithelial cells. Nishii et al., (2013) detected a mild increase in HIF-1? and VEGF levels, and increased angiogenesis in BHD-lung cysts but not control tissues. As there is no evidence of second hit mutations in lung tissue any changes are more likely due to haploinsufficiency that a complete loss of function. The resulting milder changes in HIF activity could explain the lack of tumourigenic cell proliferation in pulmonary cysts.

Aberrant HIF activity in renal tumours suggests an important role in tumourigenesis. Increased understanding of the expression and targets of HIF-? isoforms under normal and renal tumourigenic conditions will enable identification of new drug targets. In addition, as HIF signalling appears to play a role in other aspects of BHD pathology, such treatments could be applicable to these systems.

• Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, Zbar B. Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proc Natl Acad Sci U S A. 2006 Oct 17;103(42):15552-7 PubMed PMID: 17028174.
• Doonachar A, Gallo MD, Doukas D, Pasricha R, Lantsberg I, Schoenfeld AR. Differential effects of HIF-? isoforms on apoptosis in renal carcinoma cell lines. Cancer Cell Int. 2015 Feb 22;15:23. doi: 10.1186/s12935-015-0175-3. eCollection 2015. PubMed PMID: 25729330.
• Hudson CC, Liu M, Chiang GG, Otterness DM, Loomis DC, Kaper F, Giaccia AJ, Abraham RT. Regulation of hypoxia-inducible factor 1alpha expression and function by the mammalian target of rapamycin. Mol Cell Biol. 2002 Oct;22(20):7004-14. PubMed PMID: 12242281.
• Kondo K, Kim WY, Lechpammer M, Kaelin WG Jr. Inhibition of HIF2alpha is sufficient to suppress pVHL-defective tumor growth. PLoS Biol. 2003 Dec;1(3):E83. PubMed PMID: 14691554.
• Nishii T, Tanabe M, Tanaka R, Matsuzawa T, Okudela K, Nozawa A, Nakatani Y, Furuya M. Unique mutation, accelerated mTOR signaling and angiogenesis in the pulmonary cysts of Birt-Hogg-Dubé syndrome. Pathol Int. 2013 Jan;63(1):45-55. PubMed PMID: 23356225.
• Preston RS, Philp A, Claessens T, Gijezen L, Dydensborg AB, Dunlop EA, Harper KT, Brinkhuizen T, Menko FH, Davies DM, Land SC, Pause A, Baar K, van Steensel MA, Tee AR. Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene. 2011 Mar 10;30(10):1159-73 PubMed PMID: 21057536.
• Schoenfeld AR, Parris T, Eisenberger A, Davidowitz EJ, De Leon M, Talasazan F, Devarajan P, Burk RD. The von Hippel-Lindau tumor suppressor gene protects cells from UV-mediated apoptosis. Oncogene. 2000 Nov 30;19(51):5851-7. PubMed PMID: 11127815.
• Yan M, Gingras MC, Dunlop EA, Nouët Y, Dupuy F, Jalali Z, Possik E, Coull BJ, Kharitidi D, Dydensborg AB, Faubert B, Kamps M, Sabourin S, Preston RS, Davies DM, Roughead T, Chotard L, van Steensel MA, Jones R, Tee AR, Pause A. The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation. J Clin Invest. 2014 Jun;124(6):2640-50. PubMed PMID: 24762438.

...