Daniele M. Gilkes

Hypoxia and the extracellular matrix: drivers of tumour metastasis.

Of the deaths attributed to cancer, 90% are due to metastasis, and treatments that prevent or cure metastasis remain elusive. Emerging data indicate that hypoxia and the extracellular matrix (ECM) might have crucial roles in metastasis. During tumour evolution, changes in the composition and the overall content of the ECM reflect both its biophysical and biological properties and these strongly influence tumour and stromal cell properties, such as proliferation and motility. Originally thought of as independent contributors to metastatic spread, recent studies have established a direct link between hypoxia and the composition and the organization of the ECM, which suggests a new model in which multiple microenvironmental signals might converge to synergistically influence metastatic outcome.

Hypoxia-inducible factor 1 is a master regulator of breast cancer metastatic niche formation

Primary tumors facilitate metastasis by directing bone marrow-derived cells (BMDCs) to colonize the lungs before the arrival of cancer cells. Here, we demonstrate that hypoxia-inducible factor 1 (HIF-1) is a critical regulator of breast cancer metastatic niche formation through induction of multiple members of the lysyl oxidase (LOX) family, including LOX, LOX-like 2, and LOX-like 4, which catalyze collagen cross-linking in the lungs before BMDC recruitment. Only a subset of LOX family members was expressed in any individual breast cancer, but HIF-1 was required for expression in each case. Knockdown of HIF-1 or hypoxia-induced LOX family members reduced collagen cross-linking, CD11b+ BMDC recruitment, and metastasis formation in the lungs of mice after orthotopic transplantation of human breast cancer cells. Metastatic niche formation is an HIF-1–dependent event during breast cancer progression.

ATM and Chk2-dependent phosphorylation of MDMX contribute to p53 activation after DNA damage

The p53 tumor suppressor is activated after DNA damage to maintain genomic stability and prevent transformation. Rapid activation of p53 by ionizing radiation is dependent on signaling by the ATM kinase. MDM2 and MDMX are important p53 regulators and logical targets for stress signals. We found that DNA damage induces ATM‐dependent phosphorylation and degradation of MDMX. Phosphorylated MDMX is selectively bound and degraded by MDM2 preceding p53 accumulation and activation. Reduction of MDMX level by RNAi enhances p53 response to DNA damage. Loss of ATM prevents MDMX degradation and p53 stabilization after DNA damage. Phosphorylation of MDMX on S342, S367, and S403 were detected by mass spectrometric analysis, with the first two sites confirmed by phosphopeptide‐specific antibodies. Mutation of MDMX on S342, S367, and S403 each confers partial resistance to MDM2‐mediated ubiquitination and degradation. Phosphorylation of S342 and S367 in vivo require the Chk2 kinase. Chk2 also stimulates MDMX ubiquitination and degradation by MDM2. Therefore, the E3 ligase activity of MDM2 is redirected to MDMX after DNA damage and contributes to p53 activation.

MDMX Overexpression Prevents p53 Activation by the MDM2 Inhibitor Nutlin

The p53 tumor suppressor plays a key role in maintaining genomic stability and protection against malignant transformation. MDM2 and MDMX are both p53-binding proteins that regulate p53 stability and activity. Recent development of the MDM2 inhibitor Nutlin 3 has greatly facilitated functional analysis of MDM2-p53 binding. We found that although MDMX is homologous to MDM2 and binds to the same region on p53 N terminus, Nutlin does not disrupt p53-MDMX interaction. The ability of Nutlin to activate p53 is compromised in tumor cells overexpressing MDMX. Combination of Nutlin with MDMX siRNA resulted in synergistic activation of p53 and growth arrest. These results suggest that MDMX is also a valid target for p53 activation in tumor cells. Development of novel compounds that are MDMX-specific or optimized for dual-inhibition of MDM2 and MDMX are necessary to achieve full activation of p53 in tumor cells.

HIF-1-dependent expression of angiopoietin-like 4 and L1CAM mediates vascular metastasis of hypoxic breast cancer cells to the lungs

Most cases of breast cancer (BrCa) mortality are due to vascular metastasis. BrCa cells must intravasate through endothelial cells (ECs) to enter a blood vessel in the primary tumor and then adhere to ECs and extravasate at the metastatic site. In this study we demonstrate that inhibition of hypoxia-inducible factor (HIF) activity in BrCa cells by RNA interference or digoxin treatment inhibits primary tumor growth and also inhibits the metastasis of BrCa cells to the lungs by blocking the expression of angiopoietin-like 4 (ANGPTL4) and L1 cell adhesion molecule (L1CAM). ANGPTL4 is a secreted factor that inhibits EC–EC interaction, whereas L1CAM increases the adherence of BrCa cells to ECs. Interference with HIF, ANGPTL4 or L1CAM expression inhibits vascular metastasis of BrCa cells to the lungs.

Activation of Dendritic Cells via Inhibition of Jak2/STAT3 Signaling

Signaling via Jak2/STAT3 is critically important for normal dendritic cell (DC) differentiation. In addition, we have previously demonstrated that hyperactivation of the Jak2/STAT3 pathway induced by tumor-derived factors (TDF) may be responsible for abnormal DC differentiation in cancer. In this study, using a novel selective inhibitor of Jak2/STAT3, JSI-124, we investigated the mechanism of the Jak2/STAT3 effect on DCs and the possibility of pharmacological regulation of DC differentiation in cancer. Our experiments have demonstrated that JSI-124 overcomes the differentiation block induced by TDF and promotes the differentiation of mature DCs and macrophages. Surprisingly, inhibition of Jak2/STAT3 signaling resulted in dramatic activation of immature DCs generated in the presence of TDF as well as in control medium. This activation manifested in up-regulation of MHC class II, costimulatory molecules, and a dramatic increase in the ability to stimulate allogeneic or Ag-specific T cells. Inhibition of Jak2/STAT3 signaling resulted in activation of the transcription factor NF-κB. This up-regulation was not due to a conventional pathway involving IκBα, but was probably due to a block of the dominant negative effect of STAT3. This indicates that Jak2/STAT3 play an important role in negative regulation of DC activation, and pharmacological inhibition of the Jak2/STAT3 pathway can be used to enhance DC function.

Hypoxia-inducible Factor 1 (HIF-1) Promotes Extracellular Matrix Remodeling under Hypoxic Conditions by Inducing P4HA1, P4HA2, and PLOD2 Expression in Fibroblasts

Background: In hypoxic cells, HIF-1 transactivates genes with roles in developmental, physiological, and disease processes. Results: HIF-1 activity in hypoxic fibroblasts promotes extracellular matrix (ECM) remodeling by inducing expression of the collagen hydroxylases P4HA1, P4HA2, and PLOD2. Conclusion: HIF-1 stimulates ECM fiber alignment, which influences cell morphology, adhesion, and directional migration. Significance: Hypoxia induces changes in ECM that have non-cell-autonomous effects on cell behavior. 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.

Hypoxia-inducible factors are required for chemotherapy resistance of breast cancer stem cells

Significance Breast cancer stem cells play essential roles in tumor growth, maintenance, and recurrence after chemotherapy. We report that treatment of human breast cancer cells with chemotherapy results in an enrichment of breast cancer stem cells among the surviving cells, which is dependent upon the activity of hypoxia-inducible factors (HIFs). Studies in mouse tumor models suggest that combining chemotherapy with drugs that block HIF activity may improve the survival of breast cancer patients. Triple negative breast cancers (TNBCs) are defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 expression, and are treated with cytotoxic chemotherapy such as paclitaxel or gemcitabine, with a durable response rate of less than 20%. TNBCs are enriched for the basal subtype gene expression profile and the presence of breast cancer stem cells, which are endowed with self-renewing and tumor-initiating properties and resistance to chemotherapy. Hypoxia-inducible factors (HIFs) and their target gene products are highly active in TNBCs. Here, we demonstrate that HIF expression and transcriptional activity are induced by treatment of MDA-MB-231, SUM-149, and SUM-159, which are human TNBC cell lines, as well as MCF-7, which is an ER+/PR+ breast cancer line, with paclitaxel or gemcitabine. Chemotherapy-induced HIF activity enriched the breast cancer stem cell population through interleukin-6 and interleukin-8 signaling and increased expression of multidrug resistance 1. Coadministration of HIF inhibitors overcame the resistance of breast cancer stem cells to paclitaxel or gemcitabine, both in vitro and in vivo, leading to tumor eradication. Increased expression of HIF-1α or HIF target genes in breast cancer biopsies was associated with decreased overall survival, particularly in patients with basal subtype tumors and those treated with chemotherapy alone. Based on these results, clinical trials are warranted to test whether treatment of patients with TNBC with a combination of cytotoxic chemotherapy and HIF inhibitors will improve patient survival.

Hypoxia-inducible factor–dependent breast cancer–mesenchymal stem cell bidirectional signaling promotes metastasis

Metastasis involves critical interactions between cancer and stromal cells. Intratumoral hypoxia promotes metastasis through activation of hypoxia-inducible factors (HIFs). We demonstrate that HIFs mediate paracrine signaling between breast cancer cells (BCCs) and mesenchymal stem cells (MSCs) to promote metastasis. In a mouse orthotopic implantation model, MSCs were recruited to primary breast tumors and promoted BCC metastasis to LNs and lungs in a HIF-dependent manner. Coculture of MSCs with BCCs augmented HIF activity in BCCs. Additionally, coculture induced expression of the chemokine CXCL10 in MSCs and the cognate receptor CXCR3 in BCCs, which was augmented by hypoxia. CXCR3 expression was blocked in cocultures treated with neutralizing antibody against CXCL10. Conversely, CXCL10 expression was blocked in MSCs cocultured with BCCs that did not express CXCR3 or HIFs. MSC coculture did not enhance the metastasis of HIF-deficient BCCs. BCCs and MSCs expressed placental growth factor (PGF) and its cognate receptor VEGFR1, respectively, in a HIF-dependent manner, and CXCL10 expression 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.

Hypoxia-inducible factors and RAB22A mediate formation of microvesicles that stimulate breast cancer invasion and metastasis

Significance Cancer cells release from their cell surface membrane-lined microvesicles (MVs), which contain proteins, mRNAs, and microRNAs that can be taken up by other cells. We report that breast cancer cells exposed to decreased oxygen availability (hypoxia) increase their production of MVs, which stimulate invasion and metastasis by recipient breast cancer cells. Increased MV shedding by hypoxic cells requires expression of hypoxia-inducible factors (HIFs), which activate transcription of the RAB22A gene, and expression of the small GTPase RAB22A, which is a protein that localizes to budding MVs. Our results delineate a molecular mechanism by which hypoxia increases invasion and metastasis by stimulating MV shedding and provide further evidence that addition of HIF inhibitors to current treatment regimens may improve clinical outcome. Extracellular vesicles such as exosomes and microvesicles (MVs) are shed by cancer cells, are detected in the plasma of cancer patients, and promote cancer progression, but the molecular mechanisms regulating their production are not well understood. Intratumoral hypoxia is common in advanced breast cancers and is associated with an increased risk of metastasis and patient mortality that is mediated in part by the activation of hypoxia-inducible factors (HIFs). In this paper, we report that exposure of human breast cancer cells to hypoxia augments MV shedding that is mediated by the HIF-dependent expression of the small GTPase RAB22A, which colocalizes with budding MVs at the cell surface. Incubation of naïve breast cancer cells with MVs shed by hypoxic breast cancer cells promotes focal adhesion formation, invasion, and metastasis. In breast cancer patients, RAB22A mRNA overexpression in the primary tumor is associated with decreased overall and metastasis-free survival and, in an orthotopic mouse model, RAB22A knockdown impairs breast cancer metastasis.