Sylvain Provot

GATA3 suppresses metastasis and modulates the tumour microenvironment by regulating microRNA-29b expression

Despite advances in our understanding of breast cancer, patients with metastatic disease have poor prognoses. GATA3 is a transcription factor that specifies and maintains mammary luminal epithelial cell fate, and its expression is lost in breast cancer, correlating with a worse prognosis in human patients. Here, we show that GATA3 promotes differentiation, suppresses metastasis and alters the tumour microenvironment in breast cancer by inducing microRNA-29b (miR-29b) expression. Accordingly, miR-29b is enriched in luminal breast cancers and loss of miR-29b, even in GATA3-expressing cells, increases metastasis and promotes a mesenchymal phenotype. Mechanistically, miR-29b inhibits metastasis by targeting a network of pro-metastatic regulators involved in angiogenesis, collagen remodelling and proteolysis, including VEGFA, ANGPTL4, PDGF, LOX and MMP9, and targeting ITGA6, ITGB1 and TGFB, thereby indirectly affecting differentiation and epithelial plasticity. The discovery that a GATA3-miR-29b axis regulates the tumour microenvironment and inhibits metastasis opens up possibilities for therapeutic intervention in breast cancer.

GATA3 in development and cancer differentiation: cells GATA have it!

There is increasing evidence that the numerous mechanisms that regulate cell differentiation during normal development are also involved in tumorigenesis. In breast cancer, differentiation markers expressed by the primary tumor are routinely profiled to guide clinical decisions. Indeed, numerous studies have shown that the differentiation profile correlates with the metastatic potential of tumors. The transcription factor GATA3 has emerged recently as a strong predictor of clinical outcome in human luminal breast cancer. In the mammary gland, GATA3 is required for luminal epithelial cell differentiation and commitment, and its expression is progressively lost during luminal breast cancer progression as cancer cells acquire a stem cell‐like phenotype. Importantly, expression of GATA3 in GATA3‐negative, undifferentiated breast carcinoma cells is sufficient to induce tumor differentiation and inhibits tumor dissemination in a mouse model. These findings demonstrate the exquisite ability of a differentiation factor to affect malignant properties, and raise the possibility that GATA3 or its downstream genes could be used in treating luminal breast cancer. This review highlights our recent understanding of GATA3 in both normal mammary development and tumor differentiation. J. Cell. Physiol. 222:42–49, 2010.

Fetal growth plate: a developmental model of cellular adaptation to hypoxia.

Abstract:  Fetal growth plate chondrocyte is a unique mesenchymal tissue, as it is avascular and hypoxic. Yet, chondrocytes not only survive in this environment, but also undergo all cellular processes (proliferation, growth arrest, differentiation, etc.) required for normal endochondral bone development. A crucial mediator of the adaptive response of cells to hypoxia is a transcription factor named hypoxia‐inducible factor 1α (Hif‐1α). One target of Hif‐1α transcriptional activation is the angiogenic factor vascular endothelial growth factor (VEGF), whereas Hif‐1α accumulation is controlled by the von Hippel–Lindau (VHL) tumor suppressor, an E3‐ubiquitin ligase that induces its degradation by the proteasome. We, and others, demonstrated that each component of this pathway is a critical regulator of endochondral bone development. In particular, we previously established that Hif‐1α is a survival factor for hypoxic chondrocytes, and that it also negatively regulates cell proliferation. Interestingly, we also showed that hypoxia increases extracellular matrix accumulation in a Hif‐1α‐dependent fashion. This suggested that Hif‐1α could be critically important not only for cell survival and proliferation but also for cell differentiation. We recently demonstrated that Hif‐1α is indeed a differentiation factor since it is required in mesenchymal cells both for early chondrogenesis, and for joint development.

Interaction of HIF1α and β-catenin inhibits matrix metalloproteinase 13 expression and prevents cartilage damage in mice

Significance Hypoxia-inducible factor 1α (HIF1α) is important for cell growth and survival. It modulates Wnt signaling, regulating cell differentiation and fate. Osteoarthritis (OA) is an increasingly frequent joint disorder characterized by progressive cartilage breakdown in which Wnt/β-catenin signaling triggers matrix metalloproteinase 13 (MMP13) expression and chondrocyte catabolism. Here we demonstrate HIF1α inhibits β-catenin signaling by blocking transcription factor 4 (TCF4)–β-catenin interaction and down-regulates MMP13 expression, thereby alleviating cartilage lesions, whereas the TCF4–β-catenin signaling induces an OA phenotype in mice. In OA joints, PKF118-310, a small molecule that blocked TCF4–β-catenin interaction, significantly reduced the progression of OA cartilage lesions. Thus, blockade of TCF4–β-catenin signaling by HIF1α represents a promising strategy to prevent articular cartilage loss in OA. Low oxygen tension (hypoxia) regulates chondrocyte differentiation and metabolism. Hypoxia-inducible factor 1α (HIF1α) is a crucial hypoxic factor for chondrocyte growth and survival during development. The major metalloproteinase matrix metalloproteinase 13 (MMP13) is also associated with chondrocyte hypertrophy in adult articular cartilage, the lack of which protects from cartilage degradation and osteoarthritis (OA) in mice. MMP13 is up-regulated by the Wnt/β-catenin signaling, a pathway involved in chondrocyte catabolism and OA. We studied the role of HIF1α in regulating Wnt signaling in cartilage and OA. We used mice with conditional knockout of Hif1α (∆Hif1αchon) with joint instability. Specific loss of HIF1α exacerbated MMP13 expression and cartilage destruction. Analysis of Wnt signaling in hypoxic chondrocytes showed that HIF1α lowered transcription factor 4 (TCF4)–β-catenin transcriptional activity and inhibited MMP13 expression. Indeed, HIF1α interacting with β-catenin displaced TCF4 from MMP13 regulatory sequences. Finally, ΔHif1αchon mice with OA that were injected intraarticularly with PKF118-310, an inhibitor of TCF4–β-catenin interaction, showed less cartilage degradation and reduced MMP13 expression in cartilage. Therefore, HIF1α–β-catenin interaction is a negative regulator of Wnt signaling and MMP13 transcription, thus reducing catabolism in OA. Our study contributes to the understanding of the role of HIF1α in OA and highlights the HIF1α–β-catenin interaction, thus providing new insights into the impact of hypoxia in articular cartilage.

HIF signaling in osteoblast-lineage cells promotes systemic breast cancer growth and metastasis in mice

Significance Previous work showed that primary tumors instigate systemic macroenvironmental changes supporting cancer progression and metastasis. Here, we show that activation of HIF signaling in osteoblast-lineage cells also generates systemic changes promoting breast cancer growth and dissemination in bones and outside the skeleton. Our results indicate that loss of bone homeostasis through alterations of the bone anabolism could affect breast cancer progression and present the skeleton as an important organ of the tumor macroenvironment. They also suggest that targeting the bone microenvironment could limit systemic tumor growth and dissemination in breast cancer. Bone metastasis involves dynamic interplay between tumor cells and the local stromal environment. In bones, local hypoxia and activation of the hypoxia-inducible factor (HIF)-1α in osteoblasts are essential to maintain skeletal homeostasis. However, the role of osteoblast-specific HIF signaling in cancer metastasis is unknown. Here, we show that osteoprogenitor cells (OPCs) are located in hypoxic niches in the bone marrow and that activation of HIF signaling in these cells increases bone mass and favors breast cancer metastasis to bone locally. Remarkably, HIF signaling in osteoblast-lineage cells also promotes breast cancer growth and dissemination remotely, in the lungs and in other tissues distant from bones. Mechanistically, we found that activation of HIF signaling in OPCs increases blood levels of the chemokine C-X-C motif ligand 12 (CXCL12), which leads to a systemic increase of breast cancer cell proliferation and dissemination through direct activation of the CXCR4 receptor. Hence, our data reveal a previously unrecognized role of the hypoxic osteogenic niche in promoting tumorigenesis beyond the local bone microenvironment. They also support the concept that the skeleton is an important regulator of the systemic tumor environment.

Wdr5 is required for chick skeletal development.

Wdr5, a bone morphogenetic protein 2 (BMP‐2)–induced protein belonging to the family of the WD repeat proteins, is expressed in proliferating and hypertrophic chondrocytes of the growth plate and in osteoblasts. Although previous studies have provided insight into the mechanisms by which Wdr5 affects chondrocyte and osteoblast differentiation, whether Wdr5 is required in vivo for endochondral bone development has not been addressed. In this study, using an avian replication competent retrovirus (RCAS) system delivering Wdr5 short hairpin (sh) RNA to silence Wdr5 in the developing limb, we report that reduction of Wdr5 levels delays endochondral bone development and consequently results in shortening of the skeletal elements. Shortening of the skeletal elements was due to impaired chondrocyte maturation, evidenced by a significant reduction of Runx2, type X collagen, and osteopontin expression. A decrease in Runx2, type collagen I, and ostepontin expression in osteoblasts and a subsequent defect in mineralized bone was observed as well when Wdr5 levels were reduced. Most important, retroviral misexpression of Runx2 rescued the phenotype induced by Wdr5 shRNA. These findings suggest that during limb development, Wdr5 is required for endochondral bone formation and that Wdr5 influences this process, at least in part, by regulating Runx2 expression.

Calpain-6 controls the fate of sarcoma stem cells by promoting autophagy and preventing senescence

Sarcomas are still unsolved therapeutic challenges. Cancer stem cells are believed to contribute to sarcoma development, but lack of specific markers prevents their characterization and targeting. Here, we show that calpain-6 expression is associated with cancer stem cell features. In mouse models of bone sarcoma, calpain-6-expressing cells have unique tumor-initiating and metastatic capacities. Calpain-6 levels are especially high in tumors that have been successfully propagated in mouse to establish patient-derived xenografts. We found that calpain-6 levels are increased by hypoxia in vitro and calpain-6 is detected within hypoxic areas in tumors. Furthermore, calpain-6 expression depends on the stem cell transcription network that involves Oct4, Nanog, and Sox2 and is activated by hypoxia. Calpain-6 knockdown blocks tumor development in mouse and induces depletion of the cancer stem cell population. Data from transcriptomic analyses reveal that calpain-6 expression in sarcomas inversely correlates with senescence markers. Calpain-6 knockdown suppresses hypoxia-dependent prevention of senescence entry and also promotion of autophagic flux. Together, our results demonstrate that calpain-6 identifies sarcoma cells with stem-like properties and is a mediator of hypoxia to prevent senescence, promote autophagy, and maintain the tumor-initiating cell population. These findings open what we believe is a novel therapeutic avenue for targeting sarcoma stem cells.