Publications

Abstract Background: Hypoxia inducible factor 1 alpha (HIF-1α) is a master transcription factor involved in multiple oncogenic processes. In breast cancer, HIF-1α overexpression is associated with increased resistance to radiation therapy, chemotherapy, and inferior disease-free and overall survival. Patients who achieve a pathologic complete response (pCR) following neoadjuvant therapy have improved survival outcomes. Limited data are available regarding the association between HIF-1α expression and rates of pCR. We investigated the relationship between HIF-1α overexpression and pCR rates following neoadjuvant chemotherapy for early breast cancer. Methods: Eligible women were those with HER2-negative, stage II-III breast cancer, who received anthracycline- and taxane-based neoadjuvant chemotherapy from 2002 to 2012, and were included in an institutional review board-approved Integrated Breast Cancer Research Database at Johns Hopkins. The database includes patient age, sex, menopausal status, breast cancer diagnosis, tumor histopathology, treatment history, laboratory data, and outcomes. Both diagnostic and surgical tissue blocks were retrieved from pathology archives. Whole section slides were prepared and analyzed by immunohistochemical staining with appropriate negative and positive controls. The intensity of cells positive for HIF-1α was estimated visually by a pathologist blinded to clinical data. A semi-quantitative scoring of nucleus expression was used to score HIF-1α expression: score 0 is defined as less than 1%, 1 is defined as 1-5%, 2 as 5-20% and 3 as >20% tumor cells positive for HIF-1α. Overall tumor HIF-1α negativity was defined as (0, 1) and positivity as (2, 3). We compared baseline HIF-1α status among responders (pCR defined as no invasive tumor in the breast or lymph nodes) and non-responders (no pCR) using Fisher's exact test, and evaluate the association between baseline and surgical specimens in those who did not achieve pCR using NcNemar's test. Results: A total of 122 women meeting the eligibility criteria underwent a definitive surgical procedure following neoadjuvant chemotherapy. Of those, 50 patients had no tissue blocks available at baseline and additional 16 patients' blocks did not contain sufficient tissue for analysis. Thus, tumors from 56 women were available for analysis. Median age was 50 (range 33-78), 54% were White and 35% Black; 41% and 59% of women had triple negative and hormone receptor-positive tumors, respectively; 71% women had Ki67>30%, and 80% were node-positive. Overall pCR was observed in 12 women (21%). We did not detect a significant association between HIF-1α score on the diagnostic specimen with pCR status (p=0.627). However, a positive HIF-1α score was significantly associated with positive lymph nodes (p=0.01). We observed a significant decrease in HIF-1α score following chemotherapy (p<0.001). Conclusions: We did not observe a clear association between HIF-1α expression and response to neoadjuvant chemotherapy. However, HIF-1α expression on a diagnostic specimen was statistically higher than following chemotherapy and was associated with lymph node positivity. Our study is limited by its retrospective nature and a small sample size. Citation Format: Zhi WI, Huang C-Y, Gabrielson E, Tully E, Cimino-Mathews A, Santa-Maria C, Jeter S, Kai C, Semenza G, Stearns V. Association of overexpression of hypoxia inducible factor 1α with response to neoadjuvant chemotherapy in early stage breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-62.

Extracellular matrix (ECM) composition, organization, and compliance provide both architectural and chemical cues that modulate tissue structure and function. ECM produced by stromal fibroblasts plays a key role in breast cancer invasion and metastasis, which are also stimulated by intratumoral hypoxia. Here, we demonstrate that hypoxia-inducible factor 1 (HIF-1) is a critical regulator of ECM remodeling by fibroblasts under hypoxic conditions. HIF-1 activates expression of genes encoding collagen prolyl (P4HA1 and P4HA2) and lysyl (PLOD2) hydroxylases. P4HA1 and P4HA2 are required for collagen deposition, whereas PLOD2 is required for ECM stiffening and collagen fiber alignment. Together P4HA1, P4HA2, and PLOD2 mediate remodeling of ECM composition, alignment, and mechanical properties in response to hypoxia. HIF-1-dependent ECM remodeling by hypoxic fibroblasts induces changes in breast cancer cell morphology, adhesion, and motility that promote invasion and metastasis.

Inhibition of HIF-1alpha activity impairs gastric tumor growth, angiogenesis, and vessel maturation.

<div>Abstract<p>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 <i>PLOD1</i> and <i>PLOD2</i> genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the ECM. High <i>PLOD2</i> 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. <i>Mol Cancer Res; 11(5); 456–66. ©2013 AACR</i>.</p></div>

Abstract Breast cancers (BCs) that do not express the estrogen or progesterone receptor or human epidermal growth factor receptor 2 are known as triple negative breast cancers (TNBCs). Women with TNBC receive non-targeted chemotherapy with a durable response rate of less than 20%. BC stem cells (BCSCs) are a small subpopulation of BC cells that are characterized by the capacity for infinite self-renewal; are the only BC cells capable of forming a secondary (recurrent or metastatic) BC; and must be eliminated in order to eradicate BC. Hypoxia-inducible factors (HIFs) activate hundreds of genes in TNBCs and HIF-1α expression in the diagnostic tumor biopsy is associated with patient mortality. In this paper, we report that treatment of TNBC cells with cytotoxic chemotherapy increased HIF-1α and HIF-2α protein levels and HIF target gene expression. Chemotherapy also increased the percentage of BCSCs through pathways involving interleukin-6 (IL-6), IL-8, and multidrug resistance 1. HIF inhibitors blocked increased BCSC specification in response to cytotoxic chemotherapy and combination therapy led to tumor eradication. Increased HIF target gene expression in BC biopsies was correlated with increased mortality, especially in those patients treated with chemotherapy alone. Our results suggest that HIF-dependent BCSC enrichment provides a molecular and cellular basis for the high incidence of relapse in women with TNBC.

Abstract Purpose: Pancreatic cancer is characterized by intratumoral hypoxia, early and aggressive local invasion, and metastatic potential. Hypoxia-inducible factor-1 (HIF-1) is the major transcriptional activator of hypoxia-responsive genes and intratumoral hypoxia is associated with increased risk of metastasis. However, the behavior of the cells having HIF-1 activity during the malignant progression in pancreatic cancer has not been tested. Experimental Design: We orthotopically transplanted pancreatic cancer cells stably transfected with a HIF-1–dependent luciferase reporter gene and monitored HIF-1 activity in vivo in control and POP33-treated mice. POP33 is a novel prodrug, which has potential to increase caspase-3 activity and induce apoptosis in HIF-1–active/hypoxic cells. Results: In vivo optical imaging showed that HIF-1 activity proceeded along with local invasion, the peritoneal dissemination, and the liver metastasis. HIF-1–active hypoxic cells were selectively eradicated by POP33. Moreover, selective killing of HIF-1–active hypoxic cells significantly suppressed malignant progression, resulting in a significant improvement in survival rate. Conclusions: These results show that HIF-1–active cells constitute a large proportion of invading and metastatic cells and suggest that eradication of these cells may improve the outcome in advanced pancreatic cancer, a condition for which no effective therapy currently exists.

<p>Supplementary Table S1. Primer sequences used for qPCR.</p>

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-1 and HIF-1 subunits. O2-dependent degradation of the HIF-1 subunit is mediated by prolyl hydroxylase, von HippelLindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O2-independent degradation of HIF-1 is regulated by the competition of RACK1 and HSP90 for binding to HIF-1. RACK1 binding results in the recruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1. In this report, we show that calcineurin inhibits the ubiquitination and proteasomal degradation of HIF-1. 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-1. RACK1 binds to the catalytic domain of calcineurin and is required for HIF-1 degradation induced by the calcineurin inhibitor cyclosporine A. Elongin-C and HIF-1 each bind to RACK1 and dimerization of RACK1 is required to recruit Elongin-C to HIF1. 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-1 degradation. These results indicate that intracellular calcium levels can regulate HIF-1 expression by modulating calcineurin activity and RACK1 dimerization.

Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding glucose transporters, glycolytic enzymes, vascular endothelial growth factor, and other proteins involved in O2 homeostasis and tumor progression. The expression and transcriptional activity of the HIF-1alpha subunit is regulated by the cellular O2 concentration. We demonstrate that insulin, insulin-like growth factor (IGF)-1, and IGF-2 induce expression of HIF-1alpha, which is required for expression of genes encoding IGF-2, IGF-binding protein (IGFBP)-2 and IGFBP-3. These data provide a novel mechanism by which HIF-1alpha overexpression may occur in tumor cells and contribute to an autocrine growth factor loop.

Abstract Motivation: Hypoxia inducible factors (HIFs) are critical transcriptional regulators that play fundamental roles in adaptive responses to low oxygen, both in normal and disease conditions. Dysregulated activation of HIFs has been implicated in a variety of cancers due to their role in mediating a broad range of key processes including angiogenesis, glycolysis, cell survival, cancer stem cell specification, and metastasis. Methods: In this study, we utilized virtual and cell-based high-throughput screening approaches to identify compounds that disrupt the binding of HIF-1α and HIF-2α to HIF-1β, leading to the suppression of HIF target gene expression. Results: In syngeneic animal models, we observed significant tumor regression when HIF inhibitors are combined with immunotherapy agents in multiple tumor models. The combined treatment enhanced the infiltration of natural killer (NK) cells into the tumor microenvironment. Conclusion: This study highlights the anti-tumor effects of HIF inhibitors and the immune response resulting in sustained tumor regression. The promising outcomes warrant further exploration of these inhibitors as potential cancer treatment agents. Citation Format: Shaima Salman, Tina Y. Huang, Yousang Hwang, Anmol Kumar, Dominic Dordai, Alexander D. MacKerell, Gregg L. Semenza. Broad and synergistic anti-tumor effects of small-molecule inhibitors of hypoxia inducible factors when paired with immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A004.