Maimon E. Hubbi

Calcineurin Promotes Hypoxia-inducible Factor 1α Expression by Dephosphorylating RACK1 and Blocking RACK1 Dimerization

Oxygen homeostasis represents an essential organizing principle of metazoan evolution and biology. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of transcriptional responses to changes in O2 concentration. HIF-1 is a heterodimer of HIF-1α and HIF-1β subunits. O2-dependent degradation of the HIF-1α subunit is mediated by prolyl hydroxylase, von Hippel-Lindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O2-independent degradation of HIF-1α is regulated by the competition of RACK1 and HSP90 for binding to HIF-1α. RACK1 binding results in the recruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1α. In this report, we show that calcineurin inhibits the ubiquitination and proteasomal degradation of HIF-1α. Calcineurin is a serine/threonine phosphatase that is activated by calcium and calmodulin. The phosphatase activity of calcineurin is required for its regulation of HIF-1α. RACK1 binds to the catalytic domain of calcineurin and is required for HIF-1α degradation induced by the calcineurin inhibitor cyclosporine A. Elongin-C and HIF-1α each bind to RACK1 and dimerization of RACK1 is required to recruit Elongin-C to HIF-1α. Phosphorylation of RACK1 promotes its dimerization and dephosphorylation by calcineurin inhibits dimerization. Serine 146 within the dimerization domain is phosphorylated and mutation of serine 146 impairs RACK1 dimerization and HIF-1α degradation. These results indicate that intracellular calcium levels can regulate HIF-1α expression by modulating calcineurin activity and RACK1 dimerization.

Inhibitors of hypoxia-inducible factor 1 block breast cancer metastatic niche formation and lung metastasis.

Intratumoral hypoxia, a frequent finding in metastatic cancer, results in the activation of hypoxia-inducible factors (HIFs). HIFs are implicated in many steps of breast cancer metastasis, including metastatic niche formation through increased expression of lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) proteins, enzymes that remodel collagen at the metastatic site and recruit bone marrow-derived cells (BMDCs) to the metastatic niche. We investigated the effect of two chemically and mechanistically distinct HIF inhibitors, digoxin and acriflavine, on breast cancer metastatic niche formation. Both drugs blocked the hypoxia-induced expression of LOX and LOXL proteins, collagen cross-linking, CD11b+ BMDC recruitment, and lung metastasis in an orthotopic breast cancer model. Patients with HIF-1α-overexpressing breast cancers are at increased risk of metastasis and mortality and our results suggest that such patients may benefit from aggressive therapy that includes a HIF inhibitor.

Collagen Prolyl Hydroxylases are Essential for Breast Cancer Metastasis

The presence of hypoxia and fibrosis within the primary tumor are two major risk factors for metastasis of human breast cancer. In this study, we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced invasion and metastasis to lymph nodes and lungs. Finally, we establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies and show that ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, decreases tumor fibrosis and metastasis in a mouse model of breast cancer.

Procollagen lysyl hydroxylase 2 is essential for hypoxia-induced breast cancer metastasis

Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix (ECM) in the metastatic process may lead to the development of improved therapies to treat patients with cancer. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. We found that in hypoxic breast cancer cells, hypoxia-inducible factor 1 (HIF-1) activates transcription of the PLOD1 and PLOD2 genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the ECM. High PLOD2 expression in breast cancer biopsies is associated with increased risk of mortality. We show that PLOD2 is critical for fibrillar collagen formation by breast cancer cells, increases tumor stiffness, and is required for metastasis to lymph nodes and lungs. Mol Cancer Res; 11(5); 456–66. ©2013 AACR.

Chaperone-mediated Autophagy Targets Hypoxia-inducible Factor-1α (HIF-1α) for Lysosomal Degradation

Background: Regulation of hypoxia-inducible factor-1 (HIF-1) is focused on proteasomal degradation of the HIF-1α subunit. Results: Pharmacological and genetic approaches establish that HIF-1α binds to effectors of chaperone-mediated autophagy (CMA) and is targeted for lysosomal degradation. Conclusion: CMA targets HIF-1α for lysosomal degradation. Significance: Lysosomal degradation of HIF-1α represents a novel mechanism of HIF-1 regulation and a potential therapeutic target. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that mediates adaptive responses to hypoxia. We demonstrate that lysosomal degradation of the HIF-1α subunit by chaperone-mediated autophagy (CMA) is a major regulator of HIF-1 activity. Pharmacological inhibitors of lysosomal degradation, such as bafilomycin and chloroquine, increased HIF-1α levels and HIF-1 activity, whereas activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased HIF-1α levels and HIF-1 activity. In contrast, macroautophagy inhibitors did not increase HIF-1 activity. Transcription factor EB, a master regulator of lysosomal biogenesis, also negatively regulated HIF-1 activity. HIF-1α interacts with HSC70 and LAMP2A, which are core components of the CMA machinery. Overexpression of HSC70 or LAMP2A decreased HIF-1α protein levels, whereas knockdown had the opposite effect. Finally, hypoxia increased the transcription of genes involved in CMA and lysosomal biogenesis in cancer cells. Thus, pharmacological and genetic approaches identify CMA as a major regulator of HIF-1 activity and identify interplay between autophagy and the response to hypoxia.

Hypoxia-inducible factors mediate coordinated RhoA-ROCK1 expression and signaling in breast cancer cells

Significance Breast cancers often contain regions of reduced O2 availability, leading to increased activity of hypoxia-inducible factors (HIFs). Here, we demonstrate that HIFs activate transcription of the Rho family member RHOA and Rho kinase 1 (ROCK1) genes, leading to cytoskeletal changes that underlie the invasive cancer cell phenotype. ROCK1 is a kinase that regulates myosin light-chain activity, leading to actin-myosin contraction, which is the basis for cell movement. Coordinately increased levels of RhoA and ROCK1 mRNA in human breast cancers predicted patient mortality. These results demonstrate that a microenvironmental stimulus, hypoxia, can activate a critical signal transduction pathway, independent of genomic alterations, to drive cancer progression. Overexpression of Rho kinase 1 (ROCK1) and the G protein RhoA is implicated in breast cancer progression, but oncogenic mutations are rare, and the molecular mechanisms that underlie increased ROCK1 and RhoA expression have not been determined. RhoA-bound ROCK1 phosphorylates myosin light chain (MLC), which is required for actin-myosin contractility. RhoA also activates focal adhesion kinase (FAK) signaling. Together, these pathways are critical determinants of the motile and invasive phenotype of cancer cells. We report that hypoxia-inducible factors coordinately activate RhoA and ROCK1 expression and signaling in breast cancer cells, leading to cell and matrix contraction, focal adhesion formation, and motility through phosphorylation of MLC and FAK. Thus, intratumoral hypoxia acts as an oncogenic stimulus by triggering hypoxia-inducible factor → RhoA → ROCK1 → MLC → FAK signaling in breast cancer cells.

Sirtuin-7 Inhibits the Activity of Hypoxia-Inducible Factors

Background: The HIF-1 and HIF-2 transcription factors coordinate adaptive responses to hypoxia. Results: Sirt7 decreases HIF-1α and HIF-2α protein levels independent of its deacetylase activity. Conclusion: Sirt7 inhibits the activity of the HIF-1 and HIF-2 transcription factors. Significance: This study identifies Sirt7 as a regulator of HIF-1 and HIF-2 signaling. Hypoxia-inducible factor (HIF) 1 and HIF-2 are heterodimeric proteins composed of an oxygen-regulated HIF-1α or HIF-2α subunit, respectively, and a constitutively expressed HIF-1β subunit, which mediate adaptive transcriptional responses to hypoxia. Here, we report that Sirt7 (sirtuin-7) negatively regulates HIF-1α and HIF-2α protein levels by a mechanism that is independent of prolyl hydroxylation and that does not involve proteasomal or lysosomal degradation. The effect of Sirt7 was maintained in the presence of the sirtuin inhibitor nicotinamide and upon deletion or mutation of its deacetylase domain, indicating a non-catalytic function. Knockdown of Sirt7 led to an increase in HIF-1α and HIF-2α protein levels and an increase in HIF-1 and HIF-2 transcriptional activity. Thus, we identify a novel molecular function of Sirt7 as a negative regulator of HIF signaling.

Spermidine /spermine-N1 -acetyltransferase 2 is an essential component of the ubiquitin ligase complex that regulates hypoxia-inducible factor 1α

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that functions as a master regulator of oxygen homeostasis. The HIF-1α subunit is subjected to O2-dependent prolyl hydroxylation leading to ubiquitination by the von Hippel-Lindau protein (VHL)-Elongin C ubiquitin-ligase complex and degradation by the 26 S proteasome. In this study, we demonstrate that spermidine/spermine-N1-acetyltransferase (SSAT) 2 plays an essential role in this process. SSAT2 binds to HIF-1α, VHL, and Elongin C and promotes ubiquitination of hydroxylated HIF-1α by stabilizing the interaction of VHL and Elongin C. Multivalent interactions by SSAT2 provide a mechanism to ensure efficient complex formation, which is necessary for the extremely rapid ubiquitination and degradation of HIF-1α that is observed in oxygenated cells.

MCM proteins are negative regulators of hypoxia-inducible factor 1.

MCM proteins are components of a DNA helicase that plays an essential role in DNA replication and cell proliferation. However, MCM proteins are present in excess relative to origins of replication, suggesting they may serve other functions. Decreased proliferation is a fundamental physiological response to hypoxia in many cell types, and hypoxia-inducible factor 1 (HIF-1) has been implicated in this process. Here, we demonstrate that multiple MCM proteins bind directly to the HIF-1α subunit and synergistically inhibit HIF-1 transcriptional activity via distinct O(2)-dependent mechanisms. MCM3 inhibits transactivation domain function, whereas MCM7 enhances HIF-1α ubiquitination and proteasomal degradation. HIF-1 activity decreases when quiescent cells re-enter the cell cycle, and this effect is MCM dependent. Exposure to hypoxia leads to MCM2-7 downregulation in diverse cell types. These studies reveal a function of MCM proteins apart from their DNA helicase activity and establish a direct link between HIF-1 and the cell-cycle machinery.

A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication

In response to hypoxia, HIF-1α induces cell cycle arrest by inhibiting a helicase complex that unwinds DNA in preparation for replication. Staying Wound Up During Hypoxia Decreased oxygen availability (a condition called hypoxia) triggers various cellular responses, such as increased activity of hypoxia-inducible factor 1 (HIF-1), a transcription factor that activates genes involved in helping cells to survive oxygen deprivation. Hubbi et al. found that the HIF-1α subunit also had a nontranscriptional role in cellular responses to hypoxia. Hypoxic cells undergo cell cycle arrest, and HIF-1α altered interactions between the minichromosome maintenance (MCM) helicase, which unwinds DNA in preparation for replication, and various binding partners, leading to inhibition of MCM helicase activation and decreased DNA replication. HIF-1α mutants lacking transcriptional activity retained the ability to decrease DNA replication and to induce cell cycle arrest. HIF-1α can therefore mediate adaptive cellular responses to hypoxia through both transcriptional and nontranscriptional mechanisms. Cell cycle arrest in response to hypoxia is a fundamental physiological mechanism to maintain a balance between O2 supply and demand. Many of the cellular responses to reduced O2 availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the minichromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. Although the interaction between Cdc6 and the MCM complex increased the association of the MCM proteins with chromatin, the binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. These findings establish a transcription-independent mechanism by which the stabilization of HIF-1α leads to cell cycle arrest in response to hypoxia.