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Intratumoral hypoxia occurs in 90% of solid tumors and is associated with a poor prognosis for patients. Cancer cells respond to hypoxic microenvironments by activating the transcription factors, hypoxia-inducible factor 1 (HIF1) and HIF2. Here, we studied the unique gene expression patterns of 31 different breast cancer cell lines exposed to hypoxic conditions. The EGFR, a member of the ErbB (avian erythroblastosis oncogene B) family of receptors that play a role in cell proliferation, invasion, metastasis, and apoptosis, was induced in seven of the 31 breast cancer cell lines by hypoxia. A functional hypoxia response element (HRE) was identified, which is activated upon HIF1 binding to intron 18 of the EGFR gene in cell lines in which EGFR was induced by hypoxia. CpG methylation of the EGFR HRE prevented induction under hypoxic conditions. The HRE of EGFR was methylated in normal breast tissue and some breast cancer cell lines, and could be reversed by treatment with DNA methyltransferase inhibitors. Induction of EGFR under hypoxia led to an increase in AKT, ERK, and Rb phosphorylation as well as increased levels of cyclin D1, A, B1, and E2F, and repression of p21 in an HIF1α-dependent manner, leading to cell proliferation and migration. Also, increased EGFR expression sensitized cells to EGFR inhibitors. Collectively, our data suggest that patients with hypoxic breast tumors and hypomethylated EGFR status may benefit from EGFR inhibitors currently used in the clinic. Significance: Hypoxia sensitizes breast cancer cells to EGFR inhibitors in an HIF1α- and a methylation-specific manner, suggesting patients with hypoxic tumors may benefit from EGFR inhibitors already available in the clinic.

Breast cancer is the most diagnosed cancer in women in the world. Mebendazole (MBZ) has been demonstrated to have preclinical efficacy across multiple cancers, including glioblastoma multiforme, medulloblastoma, colon, breast, pancreatic, and thyroid cancers. MBZ was also well tolerated in a recent phase I clinical trial of adults diagnosed with glioma. The mechanisms of action reported so far for MBZ include tubulin disruption, inhibiting angiogenesis, promoting apoptosis, and maintaining stemness. To elucidate additional mechanisms of action for mebendazole (MBZ), we performed RNA sequencing of three different breast cancer cell lines treated with either MBZ or vehicle control. We compared the top genes downregulated upon MBZ treatment with expression profiles of cells treated with over 15,000 perturbagens using the clue.io online analysis tool. In addition to tubulin inhibitors, the gene expression profile that correlated most with MBZ treatment matched the profile of cells treated with known hypoxia-inducible factor (HIF-1α and -2α) inhibitors. The HIF pathway is the main driver of the cellular response to hypoxia, which occurs in solid tumors. Preclinical data support using HIF inhibitors in combination with standard of care to treat solid tumors. Therefore, we tested the hypothesis that MBZ could inhibit the hypoxia response. Using RNA sequencing and HIF-reporter assays, we demonstrate that MBZ inhibits the transcriptional activity of HIFs in breast cancer cell lines and in mouse models of breast cancer by preventing the induction of HIF-1α, HIF-2α, and HIF-1β protein under hypoxia. Taken together, our results suggest that MBZ treatment has additional therapeutic efficacy in the setting of hypoxia and warrants further consideration as a cancer therapy.

Simple Summary Intratumoral hypoxia is a negative prognostic factor in breast cancer progression and recurrence. By implementing a hypoxia fate-mapping system, we followed cells that experience intratumoral hypoxia in vivo and determined that these cells have an increased ability to metastasize compared to cells that were never exposed to hypoxia. In this work, we investigate whether cells that experienced intratumoral hypoxia are also resistant to chemotherapy. By utilizing both in vivo and ex vivo models, we conclude that metastatic cells found in the lung and liver, that were exposed to hypoxia in the primary tumor, are less sensitive to doxorubicin and paclitaxel and drive recurrence after treatment. Our studies also suggest that chemoresistance is associated with a cancer stem cell-like phenotype that is maintained in post-hypoxic cells. Abstract Hypoxia occurs in 90% of solid tumors and is associated with treatment failure, relapse, and mortality. HIF-1α signaling promotes resistance to chemotherapy in cancer cell lines and murine models via multiple mechanisms including the enrichment of breast cancer stem cells (BCSCs). In this work, we utilize a hypoxia fate-mapping system to determine whether triple-negative breast cancer (TNBC) cells that experience hypoxia in the primary tumor are resistant to chemotherapy at sites of metastasis. Using two orthotopic mouse models of TNBC, we demonstrate that cells that experience intratumoral hypoxia and metastasize to the lung and liver have decreased sensitivity to doxorubicin and paclitaxel but not cisplatin or 5-FU. Resistance to therapy leads to metastatic recurrence caused by post-hypoxic cells. We further determined that the post-hypoxic cells that metastasize are enriched in pathways related to cancer stem cell gene expression. Overall, our results show that even when hypoxic cancer cells are reoxygenated in the bloodstream they retain a hypoxia-induced cancer stem cell-like phenotype that persists and promotes resistance and eventually recurrence.

Breast cancer is the most commonly diagnosed cancer in women worldwide. Approximately one-tenth of all patients with advanced breast cancer develop brain metastases resulting in an overall survival rate of fewer than 2 years. The challenges lie in developing new approaches to treat, monitor, and prevent breast cancer brain metastasis (BCBM). This review will provide an overview of BCBM from the integrated perspective of clinicians, researchers, and patient advocates. We will summarize the current management of BCBM, including diagnosis, treatment, and monitoring. We will highlight ongoing translational research for BCBM, including clinical trials and improved detection methods that can become the mainstay for BCBM treatment if they demonstrate efficacy. We will discuss preclinical BCBM research that focuses on the intrinsic properties of breast cancer cells and the influence of the brain microenvironment. Finally, we will spotlight emerging studies and future research needs to improve survival outcomes and preserve the quality of life for patients with BCBM.

Recent advances in spatial transcriptomics (ST) enable us to measure gene expression from cancer tissues while retaining their spatial context. We present a novel bioinformatics pipeline to infer molecular changes from tumor and immune cell interactions in the tumor microenvironment (TME) from ST data. Latent space methods enable inference of biological patterns from ST without the need for spot deconvolution into cell-based spatial features. While linear latent space methods yield interpretable biological patterns, interactions between tumor and immune cells can be nonlinear. To enable comprehensive inference of the pathways in the TME, we developed novel algorithms to characterize biological patterns from ST data using linear latent space methods and further nonlinear effects from their interactions. For any given set of genes, the patternSpotter tool visualizes the spatial variation in the relative contribution of individual patterns to the aggregate expression at each location in the tumor sample. Application of this tool to latent features identified using CoGAPS non-negative matrix factorization on a Visium ST (10x Genomics) data from a lymph node with pancreatic cancer metastasis confirms its known immune cell architecture. Furthermore, we develop a patternMarker algorithm to identify sets of coexpressed genes associated with the patterns, which help us to pinpoint the underlying biological processes and cell types. Further analyzing a breast cancer sample with invasive carcinoma and multiple precursor lesions demonstrates that this approach can uncover tumor and immune regions without prior reliance on pathology annotations from H&E imaging. In this case, an ensemble-based factorization of multiple dimensions enhances our resolution of intra-tumor heterogeneity and identifies distinct hormone receptor pathways in different precursor lesions with the patternMarker algorithm. Additional latent features are associated with immune cells, revealing further heterogeneity in immune infiltration between the invasive carcinoma and distinct precursor lesions. Still, the molecular interactions resulting from this infiltration induce a further non-linear alteration to transcription not captured through the inferred latent spaces. To resolve this, we develop a further interactionMarker statistic to identify regions of inter-pattern interaction and the associated genes. We apply this approach to detect additional intra-tumor heterogeneity in immune signaling from infiltration suggestive of differences in immune attack of invasive lesions. Altogether, our pipeline for latent space analysis of ST can identify the location and context-specific molecular interactions within the TME, broadly applicable to a better understanding of the key drivers of tumorigenesis and resistance to immune attack in cancer. Citation Format: Atul Deshpande, Melanie Loth, Dimitrios Sidiropoulos, QingFeng Zhu, Genevieve Stein-O'Brien, NIkhil Rao, Cedric Uytingco, Stephen Williams, Cesar Santa-Maria, Daniele M. Gilkes, Lei Zhang, Elizabeth Jaffee, Robert Anders, Ludmila Danilova, Luciane T. Kagohara, Elana J. Fertig. Uncovering the spatial landscape of tumor-immune interactions using latent spaces from spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2130.

A solid brain tumor mass places compressive forces on adjacent normal brain tissue, and clinically presents as impaired motor performance in cancer patients, ultimately limiting their quality of life. In a recent article by Jain and colleagues (Nat. Biomed. Eng. 2019;3:230–245), the biological consequences of mechanical forces imparted by a growing tumor mass are explored in both mouse models and human brain tumors, revealing a novel opportunity for therapeutic intervention.

Metastasis is the leading cause of cancer-related deaths. Recent research has implicated tumor inflammation as a promoter of metastasis. Myeloid, lymphoid, and mesenchymal cells in the tumor microenvironment promote inflammatory signaling amongst each other and together with cancer cells to modulate sustained inflammation, which may enhance cancer invasiveness. Tumor hypoxia, a state of reduced available oxygen present in the majority of solid tumors, acts as a prognostic factor for a worse outcome and is known to have a role in tumor inflammation through the regulation of inflammatory mediator signals in both cancer and neighboring cells in the microenvironment. Multiple methods to target tumor hypoxia have been developed and tested in clinical trials, and still more are emerging as the impacts of hypoxia become better understood. These strategies include mechanistic inhibition of the hypoxia inducible factor signaling pathway and hypoxia activated pro-drugs, leading to both anti-tumor and anti-inflammatory effects. This prompts a need for further research on the prevention of hypoxia-mediated inflammation in cancer. Hypoxia-targeting strategies seem to have the most potential for therapeutic benefit when combined with traditional chemotherapy agents. This paper will serve to summarize the role of the inflammatory response in metastasis, to discuss how hypoxia can enable or enhance inflammatory signaling, and to review established and emerging strategies to target the hypoxia-inflammation-metastasis axis.

Metastasis is the leading cause of breast cancer mortality. Previous studies have implicated hypoxia-induced changes in the composition and stiffness of the extracellular matrix (ECM) in the metastatic process. Therefore, the contribution of potential ECM-binding receptors in this process was explored. Using a bioinformatics approach, the expression of all integrin receptor subunits, in two independent breast cancer patient datasets, were analyzed to determine whether integrin status correlates with a validated hypoxia-inducible gene signature. Subsequently, a large panel of breast cancer cell lines was used to validate that hypoxia induces the expression of integrins that bind to collagen (ITGA1, ITGA11, ITGB1) and fibronectin (ITGA5, ITGB1). Hypoxia-inducible factors (HIF-1 and HIF-2) are directly required for ITGA5 induction under hypoxic conditions, which leads to enhanced migration and invasion of single cells within a multicellular 3D tumor spheroid but did not affect migration in a 2D microenvironment. ITGB1 expression requires HIF-1α, but not HIF-2α, for hypoxic induction in breast cancer cells. ITGA5 (α5 subunit) is required for metastasis to lymph nodes and lungs in breast cancer models, and high ITGA5 expression in clinical biopsies is associated with an increased risk of mortality. Implications: These results reveal that targeting ITGA5 using inhibitors that are currently under consideration in clinical trials may be beneficial for patients with hypoxic tumors. Mol Cancer Res; 15(6); 723–34. ©2017 AACR.

Intratumoral hypoxia stimulates enrichment of breast cancer stem cells (BCSC), which are critical for metastasis and patient mortality. Here we report a metabolic adaptation that is required for hypoxia-induced BCSC enrichment and metastasis. Hypoxia-inducible factors coordinately regulate expression of genes encoding phosphoglycerate dehydrogenase (PHGDH) and five downstream enzymes in the serine synthesis pathway and mitochondrial one-carbon (folate) cycle. RNAi-mediated silencing of PHGDH expression in both estrogen receptor–positive and negative breast cancer cells led to decreased NADPH levels, disturbed mitochondrial redox homeostasis, and increased apoptosis, which abrogated BCSC enrichment under hypoxic conditions. PHGDH-deficient cells exhibited increased oxidant levels and apoptosis, as well as loss of BCSC enrichment, in response to treatment with carboplatin or doxorubicin. PHGDH-deficient cells were relatively weakly tumorigenic and tumors that did form were deficient in BCSCs, abolishing metastatic capacity. Our findings highlight a role for PHGDH in the formation of secondary (recurrent or metastatic) tumors, with potential implications for therapeutic targeting of advanced cancers.

Background RhoB is a Rho family GTPase that is highly homologous to RhoA and RhoC. RhoA and RhoC have been shown to promote tumor progression in many cancer types; however, a distinct role for RhoB in cancer has not been delineated. Additionally, several well-characterized studies have shown that small GTPases such as RhoA, Rac1, and Cdc42 are induced in vitro under hypoxia, but whether and how hypoxia regulates RhoB in breast cancer remains elusive. Aims To determine whether and how hypoxia regulates RhoB expression and to understand the role of RhoB in breast cancer metastasis. Methods We investigated the effects of hypoxia on the expression and activation of RhoB using real-time quantitative polymerase chain reaction and western blotting. We also examined the significance of both decreased and increased RhoB expression in breast cancer using CRISPR depletion of RhoB or a vector overexpressing RhoB in 3D in vitro migration models and in an in vivo mouse model. Results We found that hypoxia significantly upregulated RhoB mRNA and protein expression resulting in increased levels of activated RhoB. Both loss of RhoB and gain of RhoB expression led to reduced migration in a 3D collagen matrix and invasion within a multicellular 3D spheroid. We showed that neither the reduction nor overexpression of RhoB affected tumor growth in vivo. While the loss of RhoB had no effect on metastasis, RhoB overexpression led to decreased metastasis to the lungs, liver, and lymph nodes of mice. Conclusion Our results suggest that RhoB may have an important role in suppressing breast cancer metastasis.

The majority of cancer-related deaths are due to metastasis, hence improved methods to biologically and computationally model metastasis are required. Computational models rely on robust data that is machine-readable. The current methods used to model metastasis in mice involve generating primary tumors by injecting human cells into immune-compromised mice, or by examining genetically engineered mice that are pre-disposed to tumor development and that eventually metastasize. The degree of metastasis can be measured using flow cytometry, bioluminescence imaging, quantitative PCR, and/or by manually counting individual lesions from metastatic tissue sections. The aforementioned methods are time-consuming and do not provide information on size distribution or spatial localization of individual metastatic lesions. In this work, we describe and provide a MATLAB script for an image-processing based method designed to obtain quantitative data from tissue sections comprised of multiple subpopulations of disseminated cells localized at metastatic sites in vivo. We further show that this method can be easily adapted for high throughput imaging of live or fixed cells in vitro under a multitude of conditions in order to assess clonal fitness and evolution. The inherent variation in mouse studies, increasing complexity in experimental design which incorporate fate-mapping of individual cells, result in the need for a large cohort of mice to generate a robust dataset. High-throughput imaging techniques such as the one that we describe will enhance the data that can be used as input for the development of computational models aimed at modeling the metastatic process.

Intratumoral hypoxia has been associated with invasion, metastasis, and treatment failure, prompting the need for a global characterization of the response to hypoxic conditions. The current study presents the results of a large-scale RNA sequencing (RNA-seq) effort, analyzing 31 breast cancer cell lines representative of breast cancer subtypes or normal mammary epithelial (NME) cells exposed to control tissue culture conditions (20% O2) or hypoxic conditions (1% O2). The results demonstrate that NME have a stronger response to hypoxia both in terms of number of genes induced by hypoxia as well as level of expression. A conserved 42-gene hypoxia signature shared across PAM50 subtypes and genes that are exclusively upregulated in Luminal A, Luminal B, and normal-like mammary epithelial cells is identified. The 42-gene expression signature is enriched in a subset of basal-like cell lines and tumors and differentiates survival among patients with basal-like tumors. Mechanistically, the hypoxia-inducible factors (HIF-1 and/or HIF-2) mediate the conserved hypoxic response. Also, four novel hypoxia-regulated and HIF-1–responsive genes were identified as part of the conserved signature. This dataset provides a novel resource to query transcriptional changes that occur in response to hypoxia and serves as a starting point for a clinical assay to aid in stratifying patients that would benefit from hypoxia-targeted therapies, some of which are currently in clinical trials. Implications: RNA-seq of 31 breast cancer cells exposed to control or hypoxic conditions reveals a conserved genomic signature that contains novel HIF-regulated genes and is prognostic for the survival of patients with triple-negative breast cancer.

// Angela M. Jimenez Valencia 1, 2 , Pei-Hsun Wu 1, 2 , Osman N. Yogurtcu 3 , Pranay Rao 1 , Josh DiGiacomo 1 , Ines Godet 1 , Lijuan He 1, 2 , Meng-Horng Lee 1, 2 , Daniele Gilkes 1, 2 , Sean X. Sun 2, 3 , Denis Wirtz 1, 2, 4 1 Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland, 21218, USA 2 Physical Sciences-Oncology Center and Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland, 21218, USA 3 Department of Mechanical Engineering, The Johns Hopkins University, Baltimore, Maryland, 21218, USA 4 Department of Oncology and Department of Pathology, The Johns Hopkins School of Medicine, Baltimore, Maryland, 21218, USA Correspondence to: Denis Wirtz, e-mail: wirtz@jhu.edu Keywords: spatio-temporal invasion, fibrosarcoma, 3D invasion model Received: June 01, 2015 Accepted: October 20, 2015 Published: October 30, 2015 ABSTRACT The physical underpinnings of fibrosarcoma cell dissemination from a tumor in a surrounding collagen-rich matrix are poorly understood. Here we show that a tumor spheroid embedded in a 3D collagen matrix exerts large contractile forces on the matrix before invasion. Cell invasion is accompanied by complex spatially and temporally dependent patterns of cell migration within and at the surface of the spheroids that are fundamentally different from migratory patterns of individual fibrosarcoma cells homogeneously distributed in the same type of matrix. Cells display a continuous transition from a round morphology at the spheroid core, to highly aligned elongated morphology at the spheroid periphery, which depends on both β 1 -integrin-based cell-matrix adhesion and myosin II/ROCK-based cell contractility. This isotropic-to-anisotropic transition corresponds to a shift in migration, from a slow and unpolarized movement at the core, to a fast, polarized and persistent one at the periphery. Our results also show that the ensuing collective invasion of fibrosarcoma cells is induced by anisotropic contractile stresses exerted on the surrounding matrix.

// Meng-Horng Lee 1, 2 , Pei-Hsun Wu 1, 2 , Daniele Gilkes 1, 2 , Ivie Aifuwa 1, 2 , Denis Wirtz 1, 2, 3 1 Johns Hopkins Physical Sciences - Oncology Center, The Johns Hopkins University, Baltimore, Maryland 21218, USA 2 Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA 3 Departments of Pathology and Oncology and Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA Correspondence to: Denis Wirtz, e-mail: wirtz@jhu.edu Keywords: cancer invasion, cell-cell interaction, protrusion, microtubule, laminin Received: March 11, 2015 Accepted: July 24, 2015 Published: August 03, 2015 ABSTRACT Recent work suggests that the dissemination of tumor cells may occur in parallel with, and even preceed, tumor growth. The mechanism for this early invasion is largely unknown. Here, we find that mammary epithelial cells (MECs) induce neighboring breast carcinoma cells (BCCs) to cross the basement membrane by secreting soluble laminin. Laminin continuously produced by MECs induce long membrane cellular protrusions in BCCs that promote their contractility and invasion into the surrounding matrix. These protrusions depend on microtubule bundles assembled de novo through laminin-integrin β1 signaling. These results describe how non-cancerous MECs can actively participate in the invasive process of BCCs.

Hypoxia-inducible factor 1α (HIF-1α) expression is a hallmark of intratumoral hypoxia that is associated with breast cancer metastasis and patient mortality. Previously, we demonstrated that HIF-1 stimulates the expression and activity of TAZ, which is a transcriptional effector of the Hippo signaling pathway, by increasing TAZ synthesis and nuclear localization. Here, we report that direct protein-protein interaction between HIF-1α and TAZ has reciprocal effects: HIF-1α stimulates transactivation mediated by TAZ and TAZ stimulates transactivation mediated by HIF-1α. Inhibition of TAZ expression impairs the hypoxic induction of HIF-1 target genes, such as PDK1, LDHA, BNIP3 and P4HA2 in response to hypoxia, whereas inhibition of HIF-1α expression impairs TAZ-mediated transactivation of the CTGF promoter. Taken together, these results complement our previous findings and establish bidirectional crosstalk between HIF-1α and TAZ that increases their transcriptional activities in hypoxic cells.

// Lisha Xiang 1,2,3 , Daniele M. Gilkes 2,3 , Hongxia Hu 2,3 , Naoharu Takano 2,3,5 , Weibo Luo 2,3 , Haiquan Lu 2,3 , John W. Bullen 2,3 , Debangshu Samanta 2,3 , Houjie Liang 1 and Gregg L. Semenza 2,3,4 1 Department of Oncology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, China 2 Vascular Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 3 McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 4 Departments of Pediatrics, Medicine, Oncology, Radiation Oncology, and Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 5 Department of Biochemistry, School of Medicine, Keio University, Tokyo, Japan Correspondence: Gregg L. Semenza, email: // Keywords : Aldefluor assay, basal-like breast cancer, mammospheres, targeted therapy, triple-negative breast cancer Received : December 11, 2014 Accepted : December 12, 2014 Published : December 18, 2014 Abstract Intratumoral hypoxia, which is associated with breast cancer metastasis and patient mortality, increases the percentage of breast cancer stem cells (BCSCs) but the underlying molecular mechanisms have not been delineated. Here we report that hypoxia-inducible factor 1 (HIF-1) triggers the expression and activity of TAZ, a transcriptional co-activator that is required for BCSC maintenance, through two discrete mechanisms. First, HIF-1 binds directly to the WWTR1 gene and activates transcription of TAZ mRNA. Second, HIF-1 activates transcription of the SIAH1 gene, which encodes a ubiquitin protein ligase that is required for the hypoxia-induced ubiquitination and proteasome-dependent degradation of LATS2, a kinase that inhibits the nuclear localization of TAZ. Inhibition of HIF-1α, TAZ, or SIAH1 expression by short hairpin RNA blocked the enrichment of BCSCs in response to hypoxia. Human breast cancer database analysis revealed that increased expression (greater than the median) of both TAZ and HIF-1 target genes, but neither one alone, is associated with significantly increased patient mortality. Taken together, these results establish a molecular mechanism for induction of the BCSC phenotype in response to hypoxia.

Signaling through glutamate receptors has been reported in human cancers, but the molecular mechanisms are not fully delineated. We report that in hepatocellular carcinoma and clear cell renal carcinoma cells, increased activity of hypoxia-inducible factors (HIFs) due to hypoxia or VHL loss-of-function, respectively, augmented release of glutamate, which was mediated by HIF-dependent expression of the SLC1A1 and SLC1A3 genes encoding glutamate transporters. In addition, HIFs coordinately regulated expression of the GRIA2 and GRIA3 genes, which encode glutamate receptors. Binding of glutamate to its receptors activated SRC family kinases and downstream pathways, which stimulated cancer cell proliferation, apoptosis resistance, migration and invasion in different cancer cell lines. Thus, coordinate regulation of glutamate transporters and receptors by HIFs was sufficient to activate key signal transduction pathways that promote cancer progression.

Hypoxia occurs in 90% of solid tumors due to inefficient vasculature and has been demonstrated to promote a more aggressive phenotype, correlating with worse patient prognosis. Current methods to measure hypoxia in human tumors only identify cells that are hypoxic at the time the tissue is collected and do not delineate cells that were transiently exposed to hypoxia but survived and adapted. To overcome this challenge, we generated a Lox-Cre dual-vector oxygen-dependent reporter system. Under hypoxia, Cre expression leads to the cleavage of a DsRed reporter and permanent expression of a GFP reporter. We stably transduced breast cancer cell lines and demonstrated an irreversible reporter-switch that occurs when cells are exposed to 0.5% O2, the typical O2 level in human breast tumors, but does not occur under physiological conditions (8% O2). To explore the utility of our system, we embedded spheroids derived from MDA-MB-231 reporter cells in collagen and cultured them under 20% O2 conditions. A defined hypoxic core (GFP+) was visible after 15 days in culture and it correlated with hypoxic O2 measurements. We then generated orthotopic breast tumors by transplanting MDA-MB-231 reporter cells into the mammary fat pad of mice. Hypoxic cells (GFP+) were localized in the peri-necrotic region of the tumor and co-localized with hypoxyprobe and HIF1α labeling. RNA sequencing of GFP+ compared to DsRed+ tumor cells demonstrated that GFP+ cells were enriched for the hallmark signature of hypoxia. Additionally, we developed a transgenic mouse expressing the hypoxia-driven construct that was bred to a tdTomato-floxed GFP mouse. A female double fluorescent/hypoxia-reporter mouse was then bred to a male FVB/N-Tg(MMTV-PyMT) mouse in order to generate triple-transgenic mice that form breast tumors that spontaneously metastasize. We systematically studied hypoxia in spontaneous breast tumors from the development of ductal hyperplasia to late stage invasive carcinoma. Tumor hypoxia was first detected in early stage ductal carcinoma in situ (DCIS) lesions. Similarly to the spheroid model, mammary organoids derived from triple-transgenic mice tumors cultured under 20% O2 conditions also exhibited a defined hypoxic core after 15 days in culture. Both orthotopic and transgenic mouse models revealed an increased potency of post-hypoxic cells at arriving to distant sites in early stage metastasis. We utilized RNA sequencing to identify potential mechanisms of enhanced metastatic capacity. Ultimately, our system allowed the fate-map of hypoxic cells during tumor progression to metastasis. We show for the first time that 3D in vitro organoids derived from PyMT tumors develop regions of hypoxia under normal tissue culture conditions. In vivo, we show that hypoxic cells are more potent at arriving at distant sites and that they retain a different gene expression that potentially confers a metastatic advantage. Citation Format: Inês Godet, Yu Jung Shin, Julia A. Ju, Soumitra Bhoyar, I Chae Ye, Guannan Wang, Saraswati Sukumar, Daniele M. Gilkes. A novel approach to fate-map hypoxic cells during tumor progression uncovers metastatic potency of post-hypoxic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2649.

The tumor microenvironment plays a crucial role in cancer progression. In breast cancer, the extracellular matrix (ECM) is especially important as it contributes to mammographic breast density. Women with higher mammographic breast densities are at a greater risk for the development of breast cancer. Despite this, much knowledge of the ECM’s effects on breast cancer progression remains elusive. To better understand how ECM signaling affects breast cancer, we utilize fibroblast-deposited, decellularized ECM scaffolds. Scaffolds are produced for in vitro use by culturing a confluent layer of fibroblast cells over six days followed by decellularization with a detergent solution. RNA sequencing analysis of MCF7 human breast cancer cells cultured on ECM scaffolds versus uncoated dishes revealed differential expression of hundreds of genes. Most notably, genes within hallmark sets for early and late responses to estrogen were significantly enriched when cells were cultured on ECM. We confirm the expression of estrogen-responsive genes in breast cancer cells cultured on ECM even in the complete absence of estrogen, and also note that the estrogen receptor (ER) itself is downregulated at both the RNA and protein-level, consistent with results of estrogen stimulation. Treatment of breast cancer cells with ER inhibitors hydroxytamoxifen and fulvestrant reduces ECM-induced expression of estrogen-responsive genes. Likewise, culturing breast cancer cells on ECM enhances phosphorylation of Erk. Inhibition of phospho-Erk also reduces the expression of the same estrogen-responsive genes in cells cultured on ECM. Through luciferase, conditioned media, and estrogen-starvation assays, we demonstrate that ECM signaling activates estrogen receptor signaling in an estrogen-independent, nonclassical mechanism. In addition, we show that ER positive cells cultured on ECM are more resistant to ER-targeted therapeutics, but not other chemotherapeutic agents like paclitaxel or doxorubicin. Ultimately, we describe a pathway in which ECM can induce estrogen-independent activation of the ER as well as resistance to estrogen-targeted therapies in breast cancer. Clinical research into the effects of mammographic breast density beyond breast cancer predisposition is sparse, and this work provides incentive for investigators to better understand the relationship between breast fibrosis and clinical outcome. Citation Format: Josh W. DiGiacomo, Inês Godet, Michael Trautmann Rodriguez, I Chae Ye, Daniele M. Gilkes. Extracellular matrix signaling modulates estrogen receptor activity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1010.

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.

Breast cancer is a global burden with a woman's lifetime risk of developing breast cancer at 1 in 8. Although breast cancer is a disease that affects mostly women, the lifetime risk in men is about 1 in 1000. Most cases of breast cancer are associated with somatic mutations in breast cells that are acquired during a person's lifetime. In this scenario, the mutations are not inherited and they do not cluster in families. In hereditary breast cancer, the specific genetic factors involved will determine the inherited cancer risk. Inherited mutations in the BRCA1 or BRCA2 genes have been well-described, but mutations in ATM, CDH1, CHEK2, PALB2, PTEN, STK11, and TP53 also confer breast cancer risk. Understanding the functional significance of hereditary mutations has opened new paths for breast cancer prevention and is uncovering promising treatment strategies.

Most solid tumors contain regions of hypoxia in which increased cell proliferation promotes increased oxygen consumption and the condition is further exacerbated as cancer cells become localized far from a functional blood vessel, further decreasing the oxygen supply. An important mechanism that promotes cell adaptation to hypoxic conditions is the expression of hypoxia-inducible factors (HIFs). Hypoxia-inducible factors transcriptionally regulate many genes involved in the invasion and metastasis of breast cancer cells. Patients, whose primary tumor biopsies show high HIF expression levels, have a greater risk of metastasis. The current review will highlight the potential role of hypoxia in breast cancer metastasis to the bone by considering the regulation of many steps in the metastatic process that include invasion, migration, margination and extravasation, as well as homing signals and regulation of the bone microenvironment.

The p53 tumor suppressor plays a key role in protection against malignant transformation. MDM2 and MDMX are important regulators of the transcriptional activity and stability of p53 by binding to its NH2 terminus. Recent studies suggest that inhibition of both MDM2 and MDMX is necessary for robust activation of p53 in certain tumor cells. However, small-molecule MDM2 inhibitors such as Nutlin fail to inhibit MDMX despite significant homology between the two proteins. The therapeutic efficacy of such compounds may be compromised by MDMX overexpression. To evaluate the feasibility and biological effects of simultaneously disrupting p53 binding to MDM2 and MDMX, we used phage display to identify a novel peptide that can inhibit p53 interactions with MDM2 (IC50 = 10 nmol/L) and MDMX (IC50 = 100 nmol/L). Expression of a scaffold protein (thioredoxin) displaying this peptide sequence by adenovirus disrupts both MDM2 and MDMX interaction with p53, resulting in efficient p53 activation, cell cycle arrest, and apoptosis of tumor cells overexpressing MDM2 and MDMX. Intratumoral injection of the adenovirus also induces growth suppression of tumor xenografts in mice in a p53-dependent fashion. These results show the therapeutic potential of targeting both MDM2 and MDMX in cancer, and provide a novel structural motif for the design of potent p53 activators. [Cancer Res 2007;67(18):8810–7]

Cystathionine β-synthase (CBS) catalyzes metabolic reactions that convert homocysteine to cystathionine. To assess the role of CBS in human glioma, cells were stably transfected with lentiviral vectors encoding shRNA targeting CBS or a nontargeting control shRNA, and subclones were injected into immunodeficient mice. Interestingly, decreased CBS expression did not affect proliferation in vitro but decreased the latency period before rapid tumor xenograft growth after subcutaneous injection and increased tumor incidence and volume following orthotopic implantation into the caudate–putamen. In soft-agar colony formation assays, CBS knockdown subclones displayed increased anchorage-independent growth. Molecular analysis revealed that CBS knockdown subclones expressed higher basal levels of the transcriptional activator hypoxia-inducible factor 2α (HIF2α/EPAS1). HIF2α knockdown counteracted the effect of CBS knockdown on anchorage-independent growth. Bioinformatic analysis of mRNA expression data from human glioma specimens revealed a significant association between low expression of CBS mRNA and high expression of angiopoietin-like 4 (ANGPTL4) and VEGF transcripts, which are HIF2 target gene products that were also increased in CBS knockdown subclones. These results suggest that decreased CBS expression in glioma increases HIF2α protein levels and HIF2 target gene expression, which promotes glioma tumor formation. Implications: CBS loss-of-function promotes glioma growth. Mol Cancer Res; 12(10); 1398–406. ©2014 AACR.

Human breast tumors contain regions of hypoxia in which cells that are located far from a functional blood vessel have significantly reduced oxygen concentrations when compared with normal mammary tissue. Breast cancer cells adapt to hypoxic conditions by increasing levels of hypoxia-inducible factors (HIFs), which induce the expression of multiple genes involved in angiogenesis, glucose utilization, resistance to oxidative stress, cell proliferation, resistance to apoptosis, invasion and metastasis. Breast cancer patients with increased HIF expression levels in primary tumor biopsies are at increased risk of metastasis. This is an important finding since 90% of breast cancer deaths are the result of metastasis, primarily to the bone, lungs, liver, brain and regional lymph nodes. Although the prognostic significance of reduced oxygen levels in primary breast tumors of cancer patients is well recognized, the mechanisms underlying hypoxia-induced, HIF-dependent breast cancer metastasis are just beginning to be uncovered. Recent studies have implicated HIF target genes in every step of the metastatic process. Drugs, such as digoxin, show the potential therapeutic effects of blocking HIF activity by decreasing primary tumor growth, vascularization, invasion and metastasis in animal models of breast cancer.

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. Cancer Res; 73(11); 3285–96. ©2013 AACR.

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-1 alpha-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., link_to_OA_fulltext

Metastasis involves critical interactions between cancer and stromal cells. Intratumoral hypoxia promotes metastasis through activation of hypoxia-inducible factors (HIFs). We have demonstrated that HIFs mediate paracrine signaling between breast cancer cells (BCCs) and mesenchymal stem cells (MSCs) that facilitate metastasis. In an orthotopic implantation model, MSCs were recruited to primary breast tumors and promoted BCC metastasis to lymph nodes and lungs in a HIF-dependent manner. In vitro gene analysis of co-culture of MSCs with BCCs showed induced expression of the chemokine CXCL10 in MSCs and its cognate receptor CXCR3 in BCCs, which was augmented by hypoxia. Further we showed that CXCR3 expression was blocked in co-cultures treated with neutralizing antibody against CXCL10. Conversely, CXCL10 expression was blocked in MSCs co-cultured with BCCs that did not express CXCR3 or HIFs. MSC co-culture did not enhance the metastasis of CXCR3-deficient or HIF-deficient BCCs. Co-culture of MSCs with BCCs augmented HIF activity in BCCs. BCCs and MSCs expressed placental growth factor (PGF) and its cognate receptor VEGF1, respectively, in a HIF-dependent manner. The expression of CXCL10 by MSCs was dependent on PGF expression by BCCs. PGF promoted metastasis of BCCs and also facilitated homing of MSCs to tumors. Thus, HIFs mediate complex and bidirectional paracrine signaling between BCCs and MSCs that stimulates breast cancer metastasis. Citation Format: Pallavi Chaturvedi, Daniele Gilkes, Gregg Semenza. Hypoxia inducible factor-dependent breast cancer-mesenchymal stem cell bidirectional signaling promotes metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 505. doi:10.1158/1538-7445.AM2013-505

Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies to treat cancer patients. Intratumoral hypoxia is found in the majority of breast cancers and is associated with an increased risk of metastasis and patient mortality. Here 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 cancer cell invasion and metastasis to lymph nodes and lungs. We establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies and evaluate the novel use of ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, which decreases tumor fibrosis and metastasis in a mouse model of breast cancer. Citation Format: Daniele M. Gilkes, Pallavi Chaturvedi, Saumendra Bajpai, Maimon Hubbi, Denis Wirtz, Gregg Semenza. Collagen prolyl hydroxylases are essential for breast cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3937. doi:10.1158/1538-7445.AM2013-3937

Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies to treat cancer patients. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. Here 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 identify that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers resulting in enhanced cancer cell invasion and metastasis to lymph nodes and lungs. We establish the prognostic significance of collagen hydroxylase expression in human breast cancer biopsies and evaluate the novel use of ethyl 3,4-dihydroxybenzoate, a hydroxylase inhibitor, which decreases tumor fibrosis and metastasis in a mouse model of breast cancer. Citation Format: Daniele M. Gilkes, Pallavi Chaturvedi, Carmen Wong, Saumendra Bajpai, Hong Wei, Maimon Hubbi, Denis Wirtz, Gregg Semenza. Collagen hydroxylases are essential for breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A3.

Increased glucose uptake and metabolism is a universal characteristic of advanced solid cancers. There are two well-established mechanisms underlying the reprogramming of tumor metabolism. First, intratumoral hypoxia induces the activity of the transcriptional activator hypoxia-inducible factor 1 (HIF-1) by inhibiting the O2-dependent prolyl and asparaginyl hydroxylases that inhibit HIF-1α stability and transactivation, respectively. Second, genetic alterations increase the activity of HIF-1, thereby increasing the expression of glucose transporters (GLUT1, GLUT3), glycolytic enzymes (ALDOA, ENO1, HK2, LDHA, PKM2), pH regulators (CAR9, NHE1, MCT4), and proteins that inhibit mitochondrial metabolism (BNIP3, PDK1). Metabolites, such as the glycolytic end-product lactate, also induce HIF-1 activity, thereby providing a signal to further increase glycolytic metabolism. Recently, we have identified a novel feed-forward mechanism by which glycolytic enzyme expression leads to increased HIF-1 transcriptional activity. Pyruvate kinase isoforms PKM1 and PKM2 are alternatively spliced products of the PKM2 gene. PKM2, but not PKM1, alters glucose metabolism in cancer cells and contributes to tumorigenesis by mechanisms that are not explained by its known biochemical activity. We show that PKM2 gene transcription is activated by HIF-1. PKM2 interacts directly with the HIF-1α subunit and promotes transactivation of HIF-1 target genes by enhancing HIF-1 binding and p300 recruitment to hypoxia response elements, whereas PKM1 fails to regulate HIF-1 activity. Interaction of PKM2 with prolyl hydroxylase 3 (PHD3) enhances PKM2 binding to HIF-1α and PKM2 coactivator function. Mass spectrometry and anti-hydroxyproline antibody assays demonstrate PKM2 hydroxylation on proline-403/408. PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O2 consumption in cancer cells. Thus, PKM2 participates in a positive feedback loop that promotes HIF-1 transactivation and reprograms glucose metabolism in cancer cells (1). HIF-1 also plays critical roles in breast cancer metastasis. HIF-1 controls metastatic niche formation by activating transcription of genes encoding lysyl oxidase (LOX) and LOX-like proteins 2 and 4, which remodel collagen in the lungs, thereby recruiting bone marrow-derived cells that establish a microenvironment suitable for colonization by breast cancer cells (2). HIF-1 also promotes the extravasation of circulating breast cancer cells in the lungs by activating transcription of the genes encoding L1CAM, which encodes a cell adhesion molecule that promotes the interaction of breast cancer cells with vascular endothelial cells (ECs), and angiopoietin-like 4, which encodes a secreted factor that inhibits EC-EC interaction (3). Inhibition of HIF-1 activity by genetic or pharmacologic strategies dramatically inhibits the metastasis of breast cancer cells to the lungs in orthotopic mouse models (2, 3). (1) Luo W. et al. Cell 2011;145:732. (2) Wong CC et al. Proc Natl Acad Sci USA 2011 Sept 12. doi:10.1073/pnas.1113483108. (3) Zhang H. et al. Oncogene 2011 Aug 22. doi: 10.1038/onc.2011.365. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr MS3-2.

An increasing number of studies have reported that primary tumors facilitate metastasis by directing bone marrow cells (BMC) to prime metastatic sites. Intratumoral hypoxia promotes metastasis through the stabilization of hypoxia-inducible factors (HIF1α and HIF2α), which upregulate a variety of genes crucial to cancer progression. It has been recently reported that hypoxic tumors induce lysyl oxidase (LOX), an amine oxidase that cross-links collagen and elastin in extracellular matrices, which is fundamental to metastatic niche formation. There are 4 other highly conserved members of the LOX family: lysyl oxidase like 1-4 (LOXL1-4). We found that different LOX members were induced by hypoxia in different human breast cancer cell lines, consistent with the unique expression pattern of LOX members in individual breast cancer patients. LOX and LOXL4 were markedly upregulated by hypoxia in MDA-MB-231 cells whereas LOXL2 was upregulated by hypoxia in MDA-MB-435 cells at the mRNA and protein levels. Hypoxic induction of various LOX members was abolished when HIF is genetically silenced or pharmacologically inhibited by HIF inhibitors acriflavine or digoxin (DIG). By microrheology and confocal imaging of collagen, we found that conditioned medium from hypoxic breast cancer cells increased collagen crosslinking compared to non-hypoxic conditioned medium. This effect was lost when HIF is knocked down. Also, we found an increase of BMC invasion through matrigel pretreated with conditioned medium from hypoxic MDA-MB-231 and MDA-MB-435 cells as compared to non-hypoxic cells. This induction was abrogated when HIF was genetically or pharmacologically inhibited. Importantly, pharmacologic (daily DIG intraperitoneal injection) or genetic inhibition of HIF in breast cancer cells significantly reduced lung metastasis and recruitment of CD11b+ BMCs to the lungs in an orthotopic implantation model. Fewer cross-linked collagen fibers were observed in the lungs of tumor-bearing mice when HIF was inhibited. Knockdown of LOX or LOXL4 in MDA-MB-231 and knockdown of LOXL2 in MDA-MB-435 cells recapitulated the results from HIF inhibition. Knockdown of LOX members in the cell lines suppressed hypoxia-induced collagen remodeling, BMC invasion in vitro, and CD11b+ recruitment to lungs in vivo. In conclusion, HIF induced secretion of multiple LOX members by primary breast cancers induces collagen remodeling and facilitates BMC recruitment to the lungs, leading to increased metastasis. Antibodies or small molecule inhibitors of LOX do not inhibit all family members. In contrast, HIF, the key determinant of LOX family expression, serves as an attractive chemotherapeutic target to inhibit niche formation and subsequent metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1521. doi:10.1158/1538-7445.AM2011-1521