Debbie Liao

Hypoxia: A key regulator of angiogenesis in cancer

Angiogenesis is an important mediator of tumor progression. As tumors expand, diffusion distances from the existing vascular supply increases resulting in hypoxia. Sustained expansion of a tumor mass requires new blood vessel formation to provide rapidly proliferating tumor cells with an adequate supply of oxygen and metabolites. The key regulator of hypoxia-induced angiogenesis is the transcription factor hypoxia inducible factor (HIF)-1. Multiple HIF-1 target genes have been shown to modulate angiogenesis by promoting the mitogenic and migratory activities of endothelial cells. Because of this, hypoxia-induced angiogenesis has become an attractive target for cancer therapy, however the mechanisms involved during this process and how best to target it for cancer therapy are still under investigation. This review will cover the current understanding of hypoxia-induced tumor angiogenesis and discuss the caveats of hypoxia-targeted antiangiogenic therapy for the treatment of cancer.

Macrophage Expression of Hypoxia-Inducible Factor-1α Suppresses T-Cell Function and Promotes Tumor Progression

T cells can inhibit tumor growth, but their function in the tumor microenvironment is often suppressed. Many solid tumors exhibit abundant macrophage infiltration and low oxygen tension, yet how hypoxic conditions may affect innate immune cells and their role in tumor progression is poorly understood. Targeted deletion of the hypoxia-responsive transcription factor hypoxia-inducible factor-1α (HIF-1α) in macrophages in a progressive murine model of breast cancer resulted in reduced tumor growth, although vascular endothelial growth factor-A levels and vascularization were unchanged. Tumor-associated macrophages can suppress tumor-infiltrating T cells by several mechanisms, and we found that hypoxia powerfully augmented macrophage-mediated T-cell suppression in vitro in a manner dependent on macrophage expression of HIF-1α. Our findings link the innate immune hypoxic response to tumor progression through induction of T-cell suppression in the tumor microenvironment.

Hypoxia-Inducible Factor-1α Is a Key Regulator of Metastasis in a Transgenic Model of Cancer Initiation and Progression

Adaptation to hypoxia is a critical step in tumor progression and is, in part, regulated by the transcription factor hypoxiainducible factor-1A (HIF-1A). Xenograft models have been extensively used to characterize the role of HIF-1A in experimental cancers. Although these models provide an understanding of tumor growth at terminal stages of malignancy, they do not address tumor initiation or metastatic progression. To elucidate these roles, HIF-1A was conditionally deleted in the mammary epithelium of a transgenic mouse model for metastatic breast cancer. Conditional deletion of HIF-1A in the mammary epithelium resulted in delayed tumor onset and retarded tumor growth; this was correlated with decreased tumor cell proliferation. Tumors with conditional deletion of HIF-1A were also less vascular during early tumor progression. Perhaps most surprisingly, deletion of HIF-1A in the mammary epithelium resulted in decreased pulmonary metastasis. These results show that whereas HIF-1A is not required for the initiation of breast tumor growth or tumor cell metastasis, the transcriptional activity of HIF-1A is a significant positive regulator of tumor progression and metastatic potential. [Cancer Res 2007;67(2):563–72]

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

Background Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer. Methodology/Principal Findings We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8+ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression. Conclusions/Significance Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.

Downregulation of transcription factor SOX2 in cancer stem cells suppresses growth and metastasis of lung cancer

Background:The cancer stem cell hypothesis suggests that neoplastic clones are maintained exclusively by a small subpopulation of cells, which have indefinite proliferation and differentiation potentials and give rise to phenotypically diverse cancer cells. Cancer stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the ‘side population’ (SP).Methods and results:The Hoechst efflux assay was used to isolate and characterize the SP from murine D121 lung carcinoma cells. Here, we demonstrated that D121-SP cells contain cancer stem cell characteristics, that is, upregulation of the transcription factors SOX2 and Oct 4 in D121-SP cells. In addition, the migration of D121-SP was decreased, and apoptosis of D121-SP was upregulated following knocking down of SOX2 in D121 cells. Importantly, downregulation of SOX2 in D121 cells markedly suppressed their metastatic potential in syngeneic mice.Conclusions:These results suggest that the SP is an enriched source of lung tumour cells with stem cell properties and that SOX2 has an important role in maintaining stem cell properties and functions that may be a potential target for effective lung cancer therapy.

Tumor‐Associated Macrophages Regulate Murine Breast Cancer Stem Cells Through a Novel Paracrine EGFR/Stat3/Sox‐2 Signaling Pathway

The cancer stem cell (CSC) hypothesis has gained significant recognition as a descriptor of tumorigenesis. Additionally, tumor‐associated macrophages (TAMs) are known to promote growth and metastasis of breast cancer. However, it is not known whether TAMs mediate tumorigenesis through regulation of breast CSCs. Here, we report that TAMs promote CSC‐like phenotypes in murine breast cancer cells by upregulating their expression of Sox‐2. These CSC‐like phenotypes were characterized by increased Sox‐2, Oct‐4, Nanog, AbcG2, and Sca‐1 gene expression, in addition to increased drug‐efflux capacity, resistance to chemotherapy, and increased tumorigenicity in vivo. Downregulation of Sox‐2 in tumor cells by siRNA blocked the ability of TAMs to induce these CSC‐like phenotypes and inhibited tumor growth in vivo. Furthermore, we identified a novel epidermal growth factor receptor (EGFR)/signal transducers and activators of transcription 3 (Stat3)/Sox‐2 paracrine signaling pathway between macrophages and mouse breast cancer cells that is required for macrophage‐induced upregulation of Sox‐2 and CSC phenotypes in tumor cells. We showed that this crosstalk was effectively blocked by the small molecule inhibitors AG1478 or CDDO‐Im against EGFR and Stat3, respectively. Therefore, our report identifies a novel role for TAMs in breast CSC regulation and establishes a rationale for targeting the EGFR/Stat3/Sox‐2 signaling pathway for CSC therapy. STEM CELLS2013;31:248–258

Endothelial Cell HIF-1α and HIF-2α Differentially Regulate Metastatic Success

Summary The hypoxia inducible transcription factors (HIFs) control many mediators of vascular response, including both angiogenic factors and small molecules such as nitric oxide (NO). In studying how endothelial HIF response itself affects metastasis, we found that loss of HIF-1α in endothelial cells reduces NO synthesis, retards tumor cell migration through endothelial layers, and restricts tumor cell metastasis, and that loss of HIF-2α has in each case the opposite effect. This results from differential regulation of NO homeostasis that in turn regulates vascular endothelial growth factor expression in an NO-dependent feedback loop. These opposing roles for the two HIF factors indicate that both they and endothelial cells regulate metastasis as malignancy progresses.

HIF1α is a critical regulator of secretory differentiation and activation, but not vascular expansion, in the mouse mammary gland

During pregnancy the mammary epithelium and its supporting vasculature rapidly expand to prepare for lactation, resulting in dramatic changes in the micro-environment. In order to investigate the role of oxygenation and metabolism in these processes, the oxygen-responsive component of the hypoxia-inducible factor (HIF) 1 complex, HIF1α, was deleted in the murine mammary gland. Although vascular density was unchanged in the HIF1α null mammary gland, loss of HIF1α impaired mammary differentiation and lipid secretion, culminating in lactation failure and striking changes in milk composition. Transplantation experiments confirmed that these developmental defects were mammary epithelial cell autonomous. These data make clear that HIF1α plays a critical role in the differentiation and function of the mammary epithelium.

The Role of proto-oncogene Fra-1 in remodeling the tumor microenvironment in support of breast tumor cell invasion and progression

A growing body of evidence indicates that interactions between neoplastic cells and tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) are crucial in promoting tumor cell invasion and progression. Macrophages have an ambiguous role in these processes as this M1 phenotype correlates with tumoricidal capacity, whereas TAMs of M2 phenotype exert tumor-promoting effects. In this study, we provide evidence that interactions between mouse breast tumor cells and TAMs remodel the TME, leading to the upregulation of Fra-1, a member of the FOS family of transcription factor. In turn, this proto-oncogene initiates activation of the IL-6/JAK/Stat3 signaling pathway. This creates a malignant switch in breast tumor cells, leading to increased release of proangiogenic factors MMP-9, vascular endothelial growth factor and transforming growth factor-β from tumor cells and intensified invasion and progression of breast cancer. Proof of the concept for the crucial role played by transcription factor Fra-1 in regulating these processes was established by specific knockdown of Fra-1 with small interfering RNA, which resulted in a marked suppression of tumor cell invasion, angiogenesis and metastasis in a mouse breast cancer model. Such a strategy could eventually lead to future efficacious treatments of metastatic breast cancer.

Targeted Therapeutic Remodeling of the Tumor Microenvironment Improves an HER-2 DNA Vaccine and Prevents Recurrence in a Murine Breast Cancer Model

The tumor microenvironment (TME) mediates immunosuppression resulting in tumor cell escape from immune surveillance and cancer vaccine failure. Immunosuppression is mediated by the STAT-3 transcription factor, which potentiates signaling in tumor and immune cells. Because immunosuppression continues to be a major inhibitor of cancer vaccine efficacy, we examined in this study whether therapeutically targeted delivery of a synthetic STAT-3 inhibitor to the TME, combined with an HER-2 DNA vaccine can improve immune surveillance against HER-2(+) breast cancer and prevent its recurrence. To this end, we developed a novel ligand-targeted nanoparticle (NP) encapsulating a CDDO-Im payload capable of specific delivery to the TME, which showed an effective therapeutic inhibition of STAT-3 activation in primary tumors. Furthermore, we showed that treatment with these NPs resulted in priming of the immune TME, characterized by increased IFN-γ, p-STAT-1, GM-CSF, IL-2, IL-15, and IL-12b and reduced TGF-β, IL-6, and IL-10 protein expression. In addition, we found significantly increased tumor infiltration by activated CD8(+) T cells, M1 macrophages, and dendritic cells. These changes correlated with delayed growth of orthotopic 4TO7 breast tumors and, when combined with an HER-2 DNA vaccine, prevented HER-2(+) primary tumor recurrence in immunocompetent mice. Furthermore, antitumor T-cell responses were enhanced in splenocytes isolated from mice treated with this combination therapy. Together, these data show effective protection from cancer recurrence through improved immune surveillance against a tumor-specific antigen.