Sébastien Tabariès

PDK1-Dependent Metabolic Reprogramming Dictates Metastatic Potential in Breast Cancer

Metabolic reprogramming is a hallmark of cellular transformation, yet little is known about metabolic changes that accompany tumor metastasis. Here we show that primary breast cancer cells display extensive metabolic heterogeneity and engage distinct metabolic programs depending on their site of metastasis. Liver-metastatic breast cancer cells exhibit a unique metabolic program compared to bone- or lung-metastatic cells, characterized by increased conversion of glucose-derived pyruvate into lactate and a concomitant reduction in mitochondrial metabolism. Liver-metastatic cells displayed increased HIF-1α activity and expression of the HIF-1α target Pyruvate dehydrogenase kinase-1 (PDK1). Silencing HIF-1α reversed the glycolytic phenotype of liver-metastatic cells, while PDK1 was specifically required for metabolic adaptation to nutrient limitation and hypoxia. Finally, we demonstrate that PDK1 is required for efficient liver metastasis, and its expression is elevated in liver metastases from breast cancer patients. Our data implicate PDK1 as a key regulator of metabolism and metastatic potential in breast cancer.

Claudin-2 Promotes Breast Cancer Liver Metastasis by Facilitating Tumor Cell Interactions with Hepatocytes

ABSTRACT We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.

Granulocytic immune infiltrates are essential for the efficient formation of breast cancer liver metastases

IntroductionBreast cancer cells display preferences for specific metastatic sites including the bone, lung and liver. Metastasis is a complex process that relies, in part, on interactions between disseminated cancer cells and resident/infiltrating stromal cells that constitute the metastatic microenvironment. Distinct immune infiltrates can either impair the metastatic process or conversely, assist in the seeding, colonization and growth of disseminated cancer cells.MethodsUsing in vivo selection approaches, we previously isolated 4T1-derived breast cancer cells that preferentially metastasize to these organs and tissues. In this study, we examined whether the propensity of breast cancer cells to metastasize to the lung, liver or bone is associated with and dependent on distinct patterns of immune cell infiltration. Immunohistocytochemistry and immunohistofluorescence approaches were used to quantify innate immune cell infiltrates within distinct metastases and depletion of Gr1+ (Ly-6C and Ly-6G) or specifically Ly-6G+ cells was performed to functionally interrogate the role of Ly-6G+ infiltrates in promoting metastasis to these organs.ResultsWe show that T lymphocytes (CD3+), myeloid-derived (Gr-1+) cells and neutrophils (Ly-6G+ or NE+) exhibit the most pronounced recruitment in lung and liver metastases, with markedly less recruitment within bone metastatic lesions. Interestingly, these infiltrating cell populations display different patterns of localization within soft tissue metastases. T lymphocytes and granulocytic immune infiltrates are localized around the periphery of liver metastases whereas they were dispersed throughout the lung metastases. Furthermore, Gr-1+ cell-depletion studies demonstrate that infiltrating myeloid-derived cells are essential for the formation of breast cancer liver metastases but dispensable for metastasis to the lung and bone. A specific role for the granulocytic component of the innate immune infiltrate was revealed through Ly-6G+ cell-depletion experiments, which resulted in significantly impaired formation of liver metastases. Finally, we demonstrate that the CD11b+/Ly-6G+ neutrophils that infiltrate and surround the liver metastases are polarized toward an N2 phenotype, which have previously been shown to enhance tumor growth and metastasis.ConclusionsOur results demonstrate that the liver-metastatic potential of breast cancer cells is heavily reliant on interactions with infiltrating Ly-6G+ cells within the liver microenvironment.

Cdx Protein Interaction with Hoxa5 Regulatory Sequences Contributes to Hoxa5 Regional Expression along the Axial Skeleton

ABSTRACT Hox gene functions are intimately linked to correct developmental expression of the genes. The identification of cis-acting regulatory sequences and their associated trans-acting factors constitutes a key step in deciphering the mechanisms underlying the correct positioning of the functional domain of Hox genes along the anterior-posterior axis. We have identified DNA elements driving Hoxa5 regionalized expression in mice, using the 2.1-kb mesodermal enhancer (MES) localized in Hoxa5 3′ flanking sequences as a starting point. The MES sequence comprises regulatory elements targeting Hoxa5 expression in the limbs, the urogenital and gastrointestinal tracts, and the cervical-upper thoracic region of the prevertebral column. A 164-bp DNA fragment within the MES caudally restricts Hoxa5 expression at the level of prevertebra 10, corresponding to the posterior limit of its functional domain. Cdx proteins directly bind to this element in vitro via two conserved sites. Preventing Cdx binding by mutating the sites causes caudal expansion of the transgene expression domain. Of all three murine Cdx proteins that bind this element in vitro, Cdx4 has emerged as a potential regional posterior repressor of Hoxa5 expression. The restrictive control provided by Cdx interactions with Hoxa5 regulatory sequences may be one of the critical events in cervicothoracic axial specification.

The IGF-Trap: Novel Inhibitor of Carcinoma Growth and Metastasis

The IGFI receptor promotes malignant progression and has been recognized as a target for cancer therapy. Clinical trials with anti-IGFIR antibodies provided evidence of therapeutic efficacy but exposed limitations due in part to effects on, and the compensatory function of, the insulin receptor system. Here, we report on the production, characterization, and biologic activity of a novel, IGF-targeting protein (the IGF-Trap) comprising a soluble form of hIGFIR and the Fc portion of hIgG1. The IGF-Trap has a high affinity for hIGFI and hIGFII but low affinity for insulin, as revealed by surface plasmon resonance. It efficiently blocked IGFIR signaling in several carcinoma cell types and inhibited tumor cell proliferation, migration, and invasion in vitro. In vivo, the IGF-Trap showed favorable pharmacokinetic properties and could suppress the growth of established breast carcinoma tumors when administered therapeutically into tumor-bearing mice, improving disease-free survival. Moreover, IGF-Trap treatment markedly reduced experimental liver metastasis of colon and lung carcinoma cells, increasing tumor cell apoptosis and reducing angiogenesis. Finally, when compared with an anti-IGFIR antibody or IGF-binding protein-1 that were used at similar or higher concentrations, the IGF-Trap showed superior therapeutic efficacy to both inhibitors. Taken together, we have developed a targeted therapeutic molecule with highly potent anticancer effects that could address limitations of current IGFIR-targeting agents. Mol Cancer Ther; 14(4); 982–93. ©2015 AACR.

Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

ABSTRACT ShcA is an important mediator of ErbB2- and transforming growth factor β (TGF-β)-induced breast cancer cell migration, invasion, and metastasis. We show that in the context of reduced ShcA levels, the bone morphogenetic protein (BMP) antagonist chordin-like 1 (Chrdl1) is upregulated in numerous breast cancer cells following TGF-β stimulation. BMPs have emerged as important modulators of breast cancer aggressiveness, and we have investigated the ability of Chrdl1 to block BMP-induced increases in breast cancer cell migration and invasion. Breast cancer-derived conditioned medium containing elevated concentrations of endogenous Chrdl1, as well as medium containing recombinant Chrdl1, suppresses BMP4-induced signaling in multiple breast cancer cell lines. Live-cell migration assays reveal that BMP4 induces breast cancer migration, which is effectively blocked by Chrdl1. We demonstrate that BMP4 also stimulated breast cancer cell invasion and matrix degradation, in part, through enhanced metalloproteinase 2 (MMP2) and MMP9 activity that is antagonized by Chrdl1. Finally, high Chrdl1 expression was associated with better clinical outcomes in patients with breast cancer. Together, our data reveal that Chrdl1 acts as a negative regulator of malignant breast cancer phenotypes through inhibition of BMP signaling.

Decreased PCSK9 expression in human hepatocellular carcinoma.

BackgroundThe management of hepatocellular carcinoma (HCC) is limited by the lack of adequate screening biomarkers and chemotherapy. In response, there has been much interest in tumor metabolism as a therapeutic target. PCSK9 stimulates internalization of the LDL-receptor, decreases cholesterol uptake into hepatocytes and affects liver regeneration. Thus, we investigated whether PCSK9 expression is altered in HCC, influencing its ability to harness cholesterol metabolism.MethodsThirty-nine patients undergoing partial hepatectomy or liver transplantation for HCC were consented for use of HCC tissue to construct a tissue microarray (TMA). The TMA was immunostained for PCSK9. Imagescope software was used to objectively determine staining, and assess for pathological and clinical correlations. PCSK9 and LDL receptor mRNA levels in flash-frozen HCC and adjacent liver tissue were determined by quantitative RT-PCR. Serum PCSK9 levels were determined by ELISA.ResultsBy immunohistochemistry, there was significantly lower expression of PCSK9 in HCC as compared to adjacent cirrhosis (p-value < 0.0001, wilcoxon signed-rank test). Significantly greater staining of PCSK9 was present in cirrhosis compared to HCC (p value <0.0001), and positivity (percentage of positive cells) was significantly greater in cirrhosis compared to HCC (p-value < 0.0001). Conversely, significantly higher expression of LDL-R was present in HCC as compared to the adjacent cirrhosis (p-value < 0.0001). There was no significant correlation of PCSK9 staining with grade of tumor, but there were significant correlations between PCSK9 staining and stage of fibrosis, according to spearman correlation test.PCSK9 mRNA levels were relatively less abundant within HCC compared to adjacent liver tissue (p-value =0.08) and normal control tissue (p-value =0.02). In contrast, serum PCSK9 levels were significantly increased among patients with HCC compared to those with chronic liver disease without HCC (p-value =0.029). LDL receptor mRNA was consistantly greater in HCC when compared to normal control tissue (p-value = 0.06) and, in general, was significantly greater in HCC when compared to adjacent liver (p-value = 0.04).ConclusionsThe decreased expression of PCSK9 and conversely increased LDL-R expression in HCC suggests that HCC modulates its local microenvironment to enable a constant energy supply. Larger-scale studies should be conducted to determine whether PCSK9 could be a therapeutic target for HCC.

Peroxiredoxin 4: A novel secreted mediator of cancer induced osteoclastogenesis

Bone is a common site of metastasis from breast and prostate carcinoma, where activation of bone resorbing osteoclasts is important for cancer progression. A large body of evidence indicates that soluble factors produced by the cancer cells act to promote osteoclast formation. Using mass spectrometry, we identified peroxiredoxin (PRDX) as a secreted mediator of cancer-induced osteoclastogenesis. Both breast (MCF7 and MDA-MB-231) and prostate (PC3 and LNCaP) carcinoma cells secreted PRDX4. PRDX4 knockdown using shRNA (shPRDX4) diminished PRDX4 secretion from MDA-MB-231 and PC3 cells and significantly decreased the ability of cancer-derived factors to induce osteoclast formation from late precursors in vitro. Tibial injection of shPRDX4 PC3 cells led to the development of significantly smaller osteolytic lesions characterized by significantly reduced osteoclast numbers compared to control PC3 cells. A meta-analysis demonstrated an increase in PRDX4 mRNA expression in carcinoma and metastatic breast and prostate tissues. Moreover, high expression of PRDX4 in the primary breast tumor was consistently associated with metastasis at 5 years. These data identify a novel function of secreted PRDX4 in mediating osteoclast activation by cancer cells.

Breast Cancer Liver Metastasis

The emergence of metastatic breast cancer is the most deadly aspect of this disease and once it has spread from the primary site, it is largely incurable. Upon dissemination from the primary tumor, breast cancer cells display preferences for specific metastatic sites. The liver represents the third most frequent site for breast cancer metastasis, following the bone and lung. Despite the evidence that hepatic metastases are associated with poor clinical outcome in breast cancer patients, little is known about the molecular mechanisms governing the spread and growth of breast cancer cells in the liver. In recent years, researchers have utilized animal model systems to isolate breast cancer cells that weakly or aggressively metastasize to the liver and have utilized gene expression profiling to compare these populations. In this manner, genes whose expression is elevated or diminished in highly metastatic breast cancer cells have been identified. We highlight both tumor intrinsic factors as well as aspects of the metastatic microenvironment that contribute to the establishment and growth of breast cancer liver metastases.

The histone H3K9 demethylase KDM3A promotes anoikis by transcriptionally activating pro-apoptotic genes BNIP3 and BNIP3L

Epithelial cells that lose attachment to the extracellular matrix undergo a specialized form of apoptosis called anoikis. Here, using large-scale RNA interference (RNAi) screening, we find that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase, plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. We find that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis we show that knockdown of Kdm3a enhances metastatic potential. Finally, we find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, our results reveal a novel transcriptional regulatory program that mediates anoikis. DOI: http://dx.doi.org/10.7554/eLife.16844.001