HIF-2 in Cancer-Associated Fibroblasts Polarizes Macrophages and Creates an Immunosuppressive Tumor Microenvironment in Pancreatic Cancer

See "Stromal HIF2 regulates immune suppression in the pancreatic cancer microenvironment," by Garcia Garcia CJ, Huang Y, Fuentes NR, et al, on page 2018.Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors, with an extremely poor prognosis. Understanding the biology of PDAC is critical for developing efficient therapies. PDAC has a unique tumor microenvironment (TME) populated by stroma rich in cancer-associated fibroblasts (CAFs), immunosuppressive myeloid-derived suppressor cells, and regulatory T cells. However, the mechanisms leading to the development of an immunosuppressive landscape in PDAC is not completely understood. Extracellular matrix and cellular components of TME are known to establish a functional firewall preventing effector immune cell infiltration, while allowing preferential entry of immunosuppressive myeloid-derived suppressor cells and regulatory T cells. Do CAFs play a role in this process? Can we eliminate CAFs to alter the immunosuppressive TME? Experimental strategies to reduce tumor stroma in mouse models using genetic and pharmacologic methods have generated opposing outcomes.1Hosein A.N. Brekken R.A. Maitra A. Pancreatic cancer stroma: an update on therapeutic targeting strategies.Nat Rev Gastroenterol Hepatol. 2020; 17: 487-505Crossref PubMed Scopus (229) Google Scholar, 2Murray E.R. Menezes S. Henry J.C. et al.Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion.Cell Rep. 2022; 38110227Abstract Full Text Full Text PDF Scopus (5) Google Scholar, 3Ozdemir B.C. Pentcheva-Hoang T. Carstens J.L. et al.Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival.Cancer Cell. 2014; 25: 719-734Abstract Full Text Full Text PDF PubMed Scopus (1401) Google Scholar, 4Olive K.P. Jacobetz M.A. Davidson C.J. et al.Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.Science. 2009; 324: 1457-1461Crossref PubMed Scopus (2389) Google Scholar, 5Chen Y. Kim J. Yang S. et al.Type I collagen deletion in alphaSMA(+) myofibroblasts augments immune suppression and accelerates progression of pancreatic cancer.Cancer Cell. 2021; 39: 548-565.e6Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar These results exemplify the dynamic role of CAFs in PDAC and suggest that perhaps modulating the function of CAFs may be a better way to tame the TME.Studies published in this issue of Gastroenterology by Garcia Garcia et al,6Garcia Garcia C.J. Huang Y. Fuentes N.R. et al.Stromal HIF2 regulates immune suppression in the pancreatic cancer microenvironment.Gastroenterology. 2022; 162: 2018-2031Abstract Full Text Full Text PDF Scopus (8) Google Scholar have shown a unique role for hypoxia-inducible factor 2 (HIF-2), a heterodimeric transcription factor in CAFs, as a key regulator of immunosuppressive TME. Previous studies have shown that stabilization (inappropriate overexpression) of HIF-2α in the pancreas impairs postnatal pancreas function with histopathologic changes similar to pancreatitis.7Flores-Martinez A. Garcia-Nunez A. Rojas A. et al.Stabilization of HIF-2alpha impacts pancreas growth.Sci Rep. 2018; 8: 13713Crossref PubMed Scopus (3) Google Scholar The present studies used a sophisticated genetic approach (dual recombinase) to delete HIF-2α in a single population of cells, fibroblasts, in a mouse model of spontaneous PDAC tumorigenesis. It is provocative to note that HIF-1α knockout (KO) in CAFs did not affect tumor growth, but HIF-2α KO in CAFs inhibited tumor growth by attenuating immunosuppressive TME. Although, mass cytometry revealed a reduced infiltration of macrophages and regulatory T cells in CAF-specific HIF-2α KO mice, HIF-2α loss in tumor cells did not affect tumor growth. These results imply that HIF-2α has cell-specific differential roles in TME.In vitro studies using HIF-2α deleted CAFs under conditions of hypoxia revealed secretion of factors inducing macrophage polarization to a M2 phenotype. Consequently, immunosuppressive TME is established (Figure 1). Is it possible then to target HIF-2 to improve immunotherapy? Systemic use of a HIF-2 inhibitor, PT2399, potentiated the therapeutic efficacy of checkpoint inhibitor blockade thereby implicating potential clinical utility of this strategy. However, not all CAFs are created equal. At least 2 subtypes of fibroblasts have been identified in PDAC. CD105 (endoglin)-positive CAFs promote tumor growth and CD105-negative CAFs suppress tumors by adaptive immunity.8Hutton C. Heider F. Blanco-Gomez A. et al.Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity.Cancer Cell. 2021; 39: 1227-1244.e20Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Future studies could reveal the biological relevance of HIFs in the functional subpopulations of CAFs and identify factors secreted by HIF-2α KO CAFs. Neutralizing the putative factors could prevent M2 polarization and reverse the immunosuppressive TME in PDAC.HIFs are evolutionarily conserved heterodimeric (α and β) transcription factors. The α subunit is regulated by oxygen. Under normoxic conditions, the α subunit is modified at 2 proline residues by prolyl hydroxylases. Enzymatic activity of prolyl hydroxylases, a dioxygenase, is dependent on oxygen, iron, and intermediates of the tricarboxylic acid cycle, such as α-ketoglutarate. Post-transcriptional modifications of HIFs target them for proteasomal degradation. Prolyl hydroxylases activity is reduced under hypoxia leading to stabilization of HIFα subunits. HIFα then dimerizes with the β subunit (aryl hydrocarbon receptor nuclear translocator) and translocates to nucleus. There are 3 HIFα subunits transcribed from distinct genes, yet all HIFs bind to an evolutionarily conserved consensus sequence, A/GCGTG, hypoxia response element. HIFs are known to interact with coactivators, such as CBP and p300, to transactivate target genes, and interactions with p300 are regulated by an inhibitory protein, CITED-2. Individual HIFs transcriptionally activate overlapping and distinct sets of genes,9Semenza G.L. The genomics and genetics of oxygen homeostasis.Annu Rev Genomics Hum Genet. 2020; 21: 183-204Crossref PubMed Scopus (36) Google Scholar but currently little is known about genes exclusively regulated by HIF-3. Because epithelial cells have a different landscape of chromatin remodeling compared with CAFs and immune cells, it is important to know the lineage-specific controls of HIF-regulated genes under hypoxic conditions. For example, detailed studies on promoter occupancy are needed to understand cell-specific role of HIFs in PDAC. Single-cell transcriptomics and metabolomics could highlight HIF-mediated changes in various cellular compartments of TME. Deletion and overexpression of HIFs have differential effects on PDAC. HIF1α KO in the pancreas in KC mice (transgenic mice expressing constitutively active Kras G12D in the pancreas) resulted in increased infiltration of a unique population of B-cells and tumor progression.10Lee K.E. Spata M. Bayne L.J. et al.Hif1a deletion reveals pro-neoplastic function of B cells in pancreatic neoplasia.Cancer Discov. 2016; 6: 256-269Crossref PubMed Scopus (134) Google Scholar In the KPC (Kras and p53) mouse model, HIF1α deletion increased invasion and metastasis.11Tiwari A. Tashiro K. Dixit A. et al.Loss of HIF1A from pancreatic cancer cells increases expression of PPP1R1B and degradation of p53 to promote invasion and metastasis.Gastroenterology. 2020; 159: 1882-1897.e5Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Also, HIF-3α overexpression in the pancreas promoted PDAC metastasis.12Zhou X. Guo X. Chen M. et al.HIF-3alpha promotes metastatic phenotypes in pancreatic cancer by transcriptional regulation of the RhoC-ROCK1 signaling pathway.Mol Cancer Res. 2018; 16: 124-134Crossref PubMed Scopus (31) Google Scholar Although the role of HIFs in tumor cells has been studied extensively, the function of individual HIFs in tumor infiltrating immune cells has not been characterized completely.In PDAC, tumor cells have distinct genetic alterations, whereas CAFs are not mutated. The fact that deletion of HIF2α in tumor cells had no impact on tumor growth means that mutational changes in cancer cells could have developed a mechanism to override the loss of function of HIF-2. One way to dissect out this issue would be to introduce constitutively active Kras G12D into the CAFs lacking HIF-2α and investigate the biological consequences on macrophage polarization. In the current studies, wild-type and HIF-2α KO CAFs were exposed to prolonged hypoxia to identify factors responsible for macrophage polarization. However, tumor hypoxia in situ is a dynamic process and is often followed by reperfusion leading to intermittent hypoxia. Transactivation by HIF was different when cells were exposed to prolonged hypoxia or short-term exposure to lower oxygen or intermittent hypoxia. Characterizing CAFs response to varying hypoxic conditions in 3-dimensional organoid models will elucidate additional pathways regulating the immunologic landscape of PDAC TME.Tumor growth and metastasis are controlled by metabolic economics. Supply and demand for oxygen and nutrients predisposes tumor cells to adapt to changing environmental conditions during tumor progression. Energy sources are often reallocated among the cells in the TME. When these changes are inadequate to sustain growth, tumor cells initiate recycling of macromolecules (autophagy) to satisfy their biosynthetic needs. HIFs are involved in each one of these processes to streamline a supply chain of metabolites. HIF-mediated utilization of glucose often leads to the accumulation of lactate under hypoxia. Lactate is then pumped out of tumor cells by monocarboxylic transporters. Extracellular lactate is taken up by CAFs and converted to pyruvate and fed back as an energy source to tumor cells.13Li F. Simon M.C. Cancer cells don't live alone: metabolic communication within tumor microenvironments.Dev Cell. 2020; 54: 183-195Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar "Metabolic symbiosis" between tumor stroma and cancer cells reduces extracellular acidosis and satisfies the energy demands. In this context, future studies on HIF-2α in CAFs could characterize metabolic cost-sharing between tumor cells and stromal cells. Because immune cells in the TME again have unique requirements for energy sources, such as glucose and/or glutamine,14Lim A.R. Rathmell W.K. Rathmell J.C. The tumor microenvironment as a metabolic barrier to effector T cells and immunotherapy.Elife. 2020; 9e55185Crossref Scopus (82) Google Scholar HIF-dependent reallocation of energy sources in TME could be exploited to enhance immune cell activation and prevent T-cell exhaustion. Investigations on the metabolic changes in the TME of HIF-2α KO CAF could answer some of these important questions. See "Stromal HIF2 regulates immune suppression in the pancreatic cancer microenvironment," by Garcia Garcia CJ, Huang Y, Fuentes NR, et al, on page 2018. See "Stromal HIF2 regulates immune suppression in the pancreatic cancer microenvironment," by Garcia Garcia CJ, Huang Y, Fuentes NR, et al, on page 2018. See "Stromal HIF2 regulates immune suppression in the pancreatic cancer microenvironment," by Garcia Garcia CJ, Huang Y, Fuentes NR, et al, on page 2018. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors, with an extremely poor prognosis. Understanding the biology of PDAC is critical for developing efficient therapies. PDAC has a unique tumor microenvironment (TME) populated by stroma rich in cancer-associated fibroblasts (CAFs), immunosuppressive myeloid-derived suppressor cells, and regulatory T cells. However, the mechanisms leading to the development of an immunosuppressive landscape in PDAC is not completely understood. Extracellular matrix and cellular components of TME are known to establish a functional firewall preventing effector immune cell infiltration, while allowing preferential entry of immunosuppressive myeloid-derived suppressor cells and regulatory T cells. Do CAFs play a role in this process? Can we eliminate CAFs to alter the immunosuppressive TME? Experimental strategies to reduce tumor stroma in mouse models using genetic and pharmacologic methods have generated opposing outcomes.1Hosein A.N. Brekken R.A. Maitra A. Pancreatic cancer stroma: an update on therapeutic targeting strategies.Nat Rev Gastroenterol Hepatol. 2020; 17: 487-505Crossref PubMed Scopus (229) Google Scholar, 2Murray E.R. Menezes S. Henry J.C. et al.Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion.Cell Rep. 2022; 38110227Abstract Full Text Full Text PDF Scopus (5) Google Scholar, 3Ozdemir B.C. Pentcheva-Hoang T. Carstens J.L. et al.Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival.Cancer Cell. 2014; 25: 719-734Abstract Full Text Full Text PDF PubMed Scopus (1401) Google Scholar, 4Olive K.P. Jacobetz M.A. Davidson C.J. et al.Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.Science. 2009; 324: 1457-1461Crossref PubMed Scopus (2389) Google Scholar, 5Chen Y. Kim J. Yang S. et al.Type I collagen deletion in alphaSMA(+) myofibroblasts augments immune suppression and accelerates progression of pancreatic cancer.Cancer Cell. 2021; 39: 548-565.e6Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar These results exemplify the dynamic role of CAFs in PDAC and suggest that perhaps modulating the function of CAFs may be a better way to tame the TME. Studies published in this issue of Gastroenterology by Garcia Garcia et al,6Garcia Garcia C.J. Huang Y. Fuentes N.R. et al.Stromal HIF2 regulates immune suppression in the pancreatic cancer microenvironment.Gastroenterology. 2022; 162: 2018-2031Abstract Full Text Full Text PDF Scopus (8) Google Scholar have shown a unique role for hypoxia-inducible factor 2 (HIF-2), a heterodimeric transcription factor in CAFs, as a key regulator of immunosuppressive TME. Previous studies have shown that stabilization (inappropriate overexpression) of HIF-2α in the pancreas impairs postnatal pancreas function with histopathologic changes similar to pancreatitis.7Flores-Martinez A. Garcia-Nunez A. Rojas A. et al.Stabilization of HIF-2alpha impacts pancreas growth.Sci Rep. 2018; 8: 13713Crossref PubMed Scopus (3) Google Scholar The present studies used a sophisticated genetic approach (dual recombinase) to delete HIF-2α in a single population of cells, fibroblasts, in a mouse model of spontaneous PDAC tumorigenesis. It is provocative to note that HIF-1α knockout (KO) in CAFs did not affect tumor growth, but HIF-2α KO in CAFs inhibited tumor growth by attenuating immunosuppressive TME. Although, mass cytometry revealed a reduced infiltration of macrophages and regulatory T cells in CAF-specific HIF-2α KO mice, HIF-2α loss in tumor cells did not affect tumor growth. These results imply that HIF-2α has cell-specific differential roles in TME. In vitro studies using HIF-2α deleted CAFs under conditions of hypoxia revealed secretion of factors inducing macrophage polarization to a M2 phenotype. Consequently, immunosuppressive TME is established (Figure 1). Is it possible then to target HIF-2 to improve immunotherapy? Systemic use of a HIF-2 inhibitor, PT2399, potentiated the therapeutic efficacy of checkpoint inhibitor blockade thereby implicating potential clinical utility of this strategy. However, not all CAFs are created equal. At least 2 subtypes of fibroblasts have been identified in PDAC. CD105 (endoglin)-positive CAFs promote tumor growth and CD105-negative CAFs suppress tumors by adaptive immunity.8Hutton C. Heider F. Blanco-Gomez A. et al.Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity.Cancer Cell. 2021; 39: 1227-1244.e20Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Future studies could reveal the biological relevance of HIFs in the functional subpopulations of CAFs and identify factors secreted by HIF-2α KO CAFs. Neutralizing the putative factors could prevent M2 polarization and reverse the immunosuppressive TME in PDAC. HIFs are evolutionarily conserved heterodimeric (α and β) transcription factors. The α subunit is regulated by oxygen. Under normoxic conditions, the α subunit is modified at 2 proline residues by prolyl hydroxylases. Enzymatic activity of prolyl hydroxylases, a dioxygenase, is dependent on oxygen, iron, and intermediates of the tricarboxylic acid cycle, such as α-ketoglutarate. Post-transcriptional modifications of HIFs target them for proteasomal degradation. Prolyl hydroxylases activity is reduced under hypoxia leading to stabilization of HIFα subunits. HIFα then dimerizes with the β subunit (aryl hydrocarbon receptor nuclear translocator) and translocates to nucleus. There are 3 HIFα subunits transcribed from distinct genes, yet all HIFs bind to an evolutionarily conserved consensus sequence, A/GCGTG, hypoxia response element. HIFs are known to interact with coactivators, such as CBP and p300, to transactivate target genes, and interactions with p300 are regulated by an inhibitory protein, CITED-2. Individual HIFs transcriptionally activate overlapping and distinct sets of genes,9Semenza G.L. The genomics and genetics of oxygen homeostasis.Annu Rev Genomics Hum Genet. 2020; 21: 183-204Crossref PubMed Scopus (36) Google Scholar but currently little is known about genes exclusively regulated by HIF-3. Because epithelial cells have a different landscape of chromatin remodeling compared with CAFs and immune cells, it is important to know the lineage-specific controls of HIF-regulated genes under hypoxic conditions. For example, detailed studies on promoter occupancy are needed to understand cell-specific role of HIFs in PDAC. Single-cell transcriptomics and metabolomics could highlight HIF-mediated changes in various cellular compartments of TME. Deletion and overexpression of HIFs have differential effects on PDAC. HIF1α KO in the pancreas in KC mice (transgenic mice expressing constitutively active Kras G12D in the pancreas) resulted in increased infiltration of a unique population of B-cells and tumor progression.10Lee K.E. Spata M. Bayne L.J. et al.Hif1a deletion reveals pro-neoplastic function of B cells in pancreatic neoplasia.Cancer Discov. 2016; 6: 256-269Crossref PubMed Scopus (134) Google Scholar In the KPC (Kras and p53) mouse model, HIF1α deletion increased invasion and metastasis.11Tiwari A. Tashiro K. Dixit A. et al.Loss of HIF1A from pancreatic cancer cells increases expression of PPP1R1B and degradation of p53 to promote invasion and metastasis.Gastroenterology. 2020; 159: 1882-1897.e5Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Also, HIF-3α overexpression in the pancreas promoted PDAC metastasis.12Zhou X. Guo X. Chen M. et al.HIF-3alpha promotes metastatic phenotypes in pancreatic cancer by transcriptional regulation of the RhoC-ROCK1 signaling pathway.Mol Cancer Res. 2018; 16: 124-134Crossref PubMed Scopus (31) Google Scholar Although the role of HIFs in tumor cells has been studied extensively, the function of individual HIFs in tumor infiltrating immune cells has not been characterized completely. In PDAC, tumor cells have distinct genetic alterations, whereas CAFs are not mutated. The fact that deletion of HIF2α in tumor cells had no impact on tumor growth means that mutational changes in cancer cells could have developed a mechanism to override the loss of function of HIF-2. One way to dissect out this issue would be to introduce constitutively active Kras G12D into the CAFs lacking HIF-2α and investigate the biological consequences on macrophage polarization. In the current studies, wild-type and HIF-2α KO CAFs were exposed to prolonged hypoxia to identify factors responsible for macrophage polarization. However, tumor hypoxia in situ is a dynamic process and is often followed by reperfusion leading to intermittent hypoxia. Transactivation by HIF was different when cells were exposed to prolonged hypoxia or short-term exposure to lower oxygen or intermittent hypoxia. Characterizing CAFs response to varying hypoxic conditions in 3-dimensional organoid models will elucidate additional pathways regulating the immunologic landscape of PDAC TME. Tumor growth and metastasis are controlled by metabolic economics. Supply and demand for oxygen and nutrients predisposes tumor cells to adapt to changing environmental conditions during tumor progression. Energy sources are often reallocated among the cells in the TME. When these changes are inadequate to sustain growth, tumor cells initiate recycling of macromolecules (autophagy) to satisfy their biosynthetic needs. HIFs are involved in each one of these processes to streamline a supply chain of metabolites. HIF-mediated utilization of glucose often leads to the accumulation of lactate under hypoxia. Lactate is then pumped out of tumor cells by monocarboxylic transporters. Extracellular lactate is taken up by CAFs and converted to pyruvate and fed back as an energy source to tumor cells.13Li F. Simon M.C. Cancer cells don't live alone: metabolic communication within tumor microenvironments.Dev Cell. 2020; 54: 183-195Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar "Metabolic symbiosis" between tumor stroma and cancer cells reduces extracellular acidosis and satisfies the energy demands. In this context, future studies on HIF-2α in CAFs could characterize metabolic cost-sharing between tumor cells and stromal cells. Because immune cells in the TME again have unique requirements for energy sources, such as glucose and/or glutamine,14Lim A.R. Rathmell W.K. Rathmell J.C. The tumor microenvironment as a metabolic barrier to effector T cells and immunotherapy.Elife. 2020; 9e55185Crossref Scopus (82) Google Scholar HIF-dependent reallocation of energy sources in TME could be exploited to enhance immune cell activation and prevent T-cell exhaustion. Investigations on the metabolic changes in the TME of HIF-2α KO CAF could answer some of these important questions. Stromal HIF2 Regulates Immune Suppression in the Pancreatic Cancer MicroenvironmentGastroenterologyVol. 162Issue 7PreviewIn this study, we show that blocking the function of a protein called hypoxia-inducible factor-2 can improve the response of pancreatic cancer to immunotherapy. Full-Text PDF Open Access