Role of VHL, HIF1A and SDH on the expression of miR-210: Implications for tumoral pseudo-hypoxic fate

Abstract

The hypoxia-inducible factor 1α (HIF-1α) and its microRNA target, miR-210, are candidate tumor-drivers of metabolic reprogramming in cancer. Neuroendocrine neoplasms such as paragangliomas (PGLs) are particularly appealing for understanding the cancer metabolic adjustments because of their associations with deregulations of metabolic enzymes, such as succinate dehydrogenase (SDH), and the von Hippel Lindau (VHL) gene involved in HIF-1α stabilization. However, the role of miR-210 in the pathogenesis of SDH-related tumors remains an unmet challenge. Herein is described an in vivo genetic analysis of the role of VHL, HIF1A and SDH on miR-210 by using knockout murine models, siRNA gene silencing, and analyses of human tumors. HIF-1α knockout abolished hypoxia-induced miR-210 expression in vivo but did not alter its constitutive expression in paraganglia. Normoxic miR-210 levels substantially increased by complete, but not partial, VHL silencing in paraganglia of knockout VHL-mice and by over-expression of p76del-mutated pVHL. Similarly, VHL-mutated PGLs, not those with decreased VHL-gene/mRNA dosage, over-expressed miR-210 and accumulate HIF-1α in most tumor cells. Ablation of SDH activity in SDHD-null cell lines or reduction of the SDHD or SDHB protein levels elicited by siRNA-induced gene silencing did not induce miR-210 whereas the presence of SDH mutations in PGLs and tumor-derived cell lines was associated with mild increase of miR-210 and the presence of a heterogeneous, HIF-1α-positive and HIF-1α-negative, tumor cell population. Thus, activation of HIF-1α is likely an early event in VHL-defective PGLs directly linked to VHL mutations, but it is a late event favored but not directly triggered by SDHx mutations. This combined analysis provides insights into the mechanisms of HIF-1α/miR-210 regulation in normal and tumor tissues potentially useful for understanding the pathogenesis of cancer and other diseases sharing similar underpinnings.

Clinical significance and peculiarities of succinate dehydrogenase B and hypoxia inducible factor 1α expression in parasympathetic versus sympathetic paragangliomas

“…The HIF‐1α protein was found to accumulate in a very small percentage of tumor cells in both types of tumors, HNPGLs and PCCs/PGLs, but the immunoscore of detectable HIF‐1α was higher in HNPGLs than in PCCs/PGLs. Furthermore, although HIF‐1α positivity in HNPGLs was significantly associated with the presence of SDHx mutations and it had a higher immunoscore in SDHB‐immunonegative than immunopositive tumors in accordance with a previous report, this association was not found in PCCs/PGLs. It should be emphasized, however, that about 47% of SDHx ‐mutated HNPGLs have <24% of positive HIF‐1α tumor cells, thus suggesting that accumulation of HIF‐1α is not an early event in the development of SDHx ‐mutated HNPGLs and that it is not a useful biomarker for the identification of SDHx ‐mutation carriers.…”

“…The VHL , NF1 , and RET genes were analyzed in blood samples from patients with clinical manifestations suggestive of inherited syndromes (n = 7) or, in the case of the RET gene, also if the patients had predominantly elevated metanephrines. Analysis of somatic VHL mutations has been previously reported for 53 HNPGLs . The VHL gene was analyzed for the presence of large deletions in tumor DNA using the multiplex ligation‐dependent probe amplification (MLPA; SALSA MLPA P016, MRC Holland) method, as recommended by the manufacturer.…”

“…The SDHB, SDHA, and HIF‐1α expressions were evaluated by immunohistochemistry in tissue sections, as previously described . A total of 146 and 75 tumor samples were available for SDHB and SDHA immunohistochemistry, respectively.…”

“…Samples with cytoplasmic punctate staining of SDHB or SDHA irrespective of the intensity were considered positive. For HIF‐1α immunohistochemistry, both the percentage of positively immunostained cells and the intensity of immunostaining were considered in scoring the samples . Staining intensity, scored from 1 to 3+, was multiplied by the percentage of stained cells to obtain an immunoscore.…”

“…Furthermore, recent microarray data have shown differences between SDHD ‐associated HNPGLs and SDHD ‐associated PCCs/PGLs, suggesting that, although both types of tumors are related to the same pathogenic gene, they develop via different mechanisms . The role of the hypoxic inducible factor 1α (HIF‐1α) on the pathogenesis of HNPGLs or PCCs/PGLs is also under debate and, although SDH deficiency has been shown to trigger HIF‐1α stabilization, the value of HIF‐1α protein as a clinical biomarker is still unknown.…”