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Supplementary Table S1. A, Patient characteristics included in the study (Fig. 7, A and B). B, Patient characteristics included in the study (Fig. 7, C and D).

Supplementary Figure S7: Recapitulated schema.

Sunitinib is an antiangiogenic therapy given as a first-line treatment for renal cell carcinoma (RCC). While treatment improves progression-free survival, most patients relapse. We hypothesized that patient relapse can stem from the development of a lymphatic network driven by the production of the main growth factor for lymphatic endothelial cells, VEGFC. In this study, we found that sunitinib can stimulate vegfc gene transcription and increase VEGFC mRNA half-life. In addition, sunitinib activated p38 MAPK, which resulted in the upregulation/activity of HuR and inactivation of tristetraprolin, two AU-rich element–binding proteins. Sunitinib stimulated a VEGFC-dependent development of lymphatic vessels in experimental tumors. This may explain our findings of increased lymph node invasion and new metastatic sites in 30% of sunitinib-treated patients and increased lymphatic vessels found in 70% of neoadjuvant treated patients. In summary, a therapy dedicated to destroying tumor blood vessels induced the development of lymphatic vessels, which may have contributed to the treatment failure. Cancer Res; 77(5); 1212–26. ©2017 AACR.

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Polo-like kinase 1 (Plk1) expression is inversely correlated with survival advantages in many cancers. However, molecular mechanisms that underlie Plk1 expression are poorly understood. Here, we uncover a hypoxia-regulated mechanism of Plk1-mediated cancer metastasis and drug resistance. We demonstrated that a HIF-2-dependent regulatory pathway drives Plk1 expression in clear cell renal cell carcinoma (ccRCC). Mechanistically, HIF-2 transcriptionally targets the hypoxia response element of the Plk1 promoter. In ccRCC patients, high expression of Plk1 was correlated to poor disease-free survival and overall survival. Loss-of-function of Plk1 in vivo markedly attenuated ccRCC growth and metastasis. High Plk1 expression conferred a resistant phenotype of ccRCC to targeted therapeutics such as sunitinib, in vitro, in vivo, and in metastatic ccRCC patients. Importantly, high Plk1 expression was defined in a subpopulation of ccRCC patients that are refractory to current therapies. Hence, we propose a therapeutic paradigm for improving outcomes of ccRCC patients., Dufies et al. find high Plk1 expression levels in aggressive clear cell renal cell carcinoma and discover that Plk1 is transcriptionally upregulated in a manner dependent on HIF-2. They also find that high Plk1 expression is correlated to a poor prognosis and resistance to tyrosine kinase inhibitor against VEGF receptor, suggesting a critical role for hypoxia/HIF-2-induced Plk1 in disease progression.

Polo-Like Kinase 1 (Plk1) expression is inversely correlated with survival advantages in many cancers. However, molecular mechanisms that underlie Plk1 expression are poorly understood. Here, we uncover a novel hypoxia-regulated mechanism of Plk1-mediated cancer metastasis and drug resistance. We demonstrated that a new HIF-2-dependent regulatory pathway drives Plk1 expression in clear cell renal cell carcinoma (ccRCC). Mechanistically, HIF-2 transcriptionally targets the hypoxia response element of the Plk1 promoter. In ccRCC patients, high expression of Plk1 was correlated to poor disease-free survival and overall survival. Loss-of-function of Plk1 in vivo markedly attenuated ccRCC growth and metastasis. High Plk1 expression conferred a resistant phenotype of ccRCC to targeted therapeutics such as sunitinib, in vitro, in vivo and in metastatic ccRCC patients. Importantly, high Plk1 expression was defined in a subpopulation of ccRCC patients that are refractory to current therapies. Hence, we propose a therapeutic paradigm for improving outcomes of ccRCC patients.

Renal cell carcinoma (RCC) still lacks prognostic and predictive biomarkers to monitor the disease and the response to therapy. The usual strategy in translational research is to start from human samples, to identify molecular markers and gene networks and then to functionally validate themin vitroand in animal models. We devised herein a completely opposite strategy from “mouse to man” by performing an aggressiveness screen and used functional genomics, imaging, clinical data and computational approaches in order to discover molecular pathways and players in renal cancer development and metastasis. Multiple cell lines for primary tumor growth, survival in the blood circulation and lung metastasis or metastatic spread from the primary tumor were generated and analyzed using a multi-layered approach which includes large-scale transcriptome, genome and methylome analyses. Transcriptome and methylome analyses demonstrated distinct clustering in three different groups. Remarkably, DNA sequencing did not show significant genomic variations in the different groups which indicates absence of clonal selection during thein vivoamplification process. Transcriptome analysis revealed distinct signatures of tumor aggressiveness which were validated in patient cohorts. Methylome analysis of full-length DNA allowed clustering of the same groups and revealed clinically relevant signatures. Furthermore, we identified SAA2 and CFB as soluble prognostic and predictive biomarkers of the therapeutic response. We also uncovered IL34 as another soluble prognostic biomarker and key regulator of renal cell carcinoma (RCC) progression. This was also functionally validatedin vivo,and a mathematical model of IL34-dependent primary tumor growth and metastasis development was provided. These results indicate that such multilayered analysis in a RCC animal model leads to meaningful results that are of translational significance.One Sentence SummaryAn aggressiveness screen with multilayer systems analysis to identify signatures and biomarkers for renal cell carcinoma aggressiveness.

The ADAMTS proteinases are a family of secreted, matrix-associated enzymes that have diverse roles in the regulation of tissue organization and vascular homeostasis. Several of the 19 human family members have been identified as having either tumor promoting or suppressing roles. We previously demonstrated that decreased ADAMTS15 expression correlated with a worse clinical outcome in mammary carcinoma (e.g., Porter et al., Int J Cancer 2006;118:1241-7). We have explored the effects of A Disintegrin and Metalloproteinase with Thrombospondin motifs-15 (ADAMTS-15) on the behavior of MDA-MB-231 and MCF-7 breast cancer cells by stable expression of either a wild-type (wt) or metalloproteinase-inactive (E362A) protein. No effects on mammary cancer cell proliferation or apoptosis were observed for either form of ADAMTS-15. However, both forms reduced cell migration on fibronectin or laminin matrices, though motility on a Type I collagen matrix was unimpaired. Knockdown of syndecan-4 attenuated the inhibitory effects of ADAMTS-15 on cell migration. In contrast to its effects on cell migration, wt ADAMTS-15 but not the E362A inactive mutant inhibited endothelial tubulogenesis in 3D collagen gels and angiogenesis in the aortic ring assay. In experimental metastasis assays in nude mice, MDA-MB-231 cells expressing either form of ADAMTS-15 showed reduced spread to the liver, though lung colonization was enhanced for cells expressing wt ADAMTS-15. These studies indicate that extracellular ADAMTS-15 has multiple actions on tumor pathophysiology. Via modulation of cell-ECM interactions, which likely involve syndecan-4, it attenuates mammary cancer cell migration independent of its metalloproteinase activity; however, its antiangiogenic action requires catalytic functionality, and its effects on metastasis in vivo are tissue niche-dependent.

Sunitinib is an antiangiogenic therapy given as a first-line treatment for renal cell carcinoma (RCC). While treatment improves progression-free survival, most patients relapse. We hypothesized that patient relapse can stem from the development of a lymphatic network driven by the production of the main growth factor for lymphatic endothelial cells, VEGFC. In this study, we found that sunitinib can stimulate vegfc gene transcription and increase VEGFC mRNA half-life. In addition, sunitinib activated p38 MAPK, which resulted in the upregulation/activity of HuR and inactivation of tristetraprolin, two AU-rich element–binding proteins. Sunitinib stimulated a VEGFC-dependent development of lymphatic vessels in experimental tumors. This may explain our findings of increased lymph node invasion and new metastatic sites in 30% of sunitinib-treated patients and increased lymphatic vessels found in 70% of neoadjuvant treated patients. In summary, a therapy dedicated to destroying tumor blood vessels induced the development of lymphatic vessels, which may have contributed to the treatment failure. Cancer Res; 77(5); 1212–26. ©2017 AACR.

Blood vascular cells and lymphatic endothelial cells (BECs and LECs, respectively) form two separate vascular systems and are functionally distinct cell types or lineages with characteristic gene expression profiles. Interconversion between these cell types has not been reported. Here, we show that in conventional in vitro angiogenesis assays, human BECs of fetal or adult origin show altered gene expression that is indicative of transition to a lymphatic-like phenotype. This change occurs in BECs undergoing tubulogenesis in fibrin, collagen or Matrigel assays, but is independent of tube formation per se, because it is not inhibited by a metalloproteinase inhibitor that blocks tubulogenesis. It is also reversible, since cells removed from 3D tubules revert to a BEC expression profile upon monolayer culture. Induction of the lymphatic-like phenotype is partially inhibited by co-culture of HUVECs with perivascular cells. These data reveal an unexpected plasticity in endothelial phenotype, which is regulated by contact with the ECM environment and/or cues from supporting cells.

The ADAMTS proteinases are a family of secreted, matrix-associated enzymes that have diverse roles in the regulation of tissue organization and vascular homeostasis. Several of the 19 human family members have been identified as having either tumor promoting or suppressing roles. We previously demonstrated that decreased ADAMTS15 expression correlated with a worse clinical outcome in mammary carcinoma (e.g., Porter et al., Int J Cancer 2006;118:1241-7). We have explored the effects of A Disintegrin and Metalloproteinase with Thrombospondin motifs-15 (ADAMTS-15) on the behavior of MDA-MB-231 and MCF-7 breast cancer cells by stable expression of either a wild-type (wt) or metalloproteinase-inactive (E362A) protein. No effects on mammary cancer cell proliferation or apoptosis were observed for either form of ADAMTS-15. However, both forms reduced cell migration on fibronectin or laminin matrices, though motility on a Type I collagen matrix was unimpaired. Knockdown of syndecan-4 attenuated the inhibitory effects of ADAMTS-15 on cell migration. In contrast to its effects on cell migration, wt ADAMTS-15 but not the E362A inactive mutant inhibited endothelial tubulogenesis in 3D collagen gels and angiogenesis in the aortic ring assay. In experimental metastasis assays in nude mice, MDA-MB-231 cells expressing either form of ADAMTS-15 showed reduced spread to the liver, though lung colonization was enhanced for cells expressing wt ADAMTS-15. These studies indicate that extracellular ADAMTS-15 has multiple actions on tumor pathophysiology. Via modulation of cell-ECM interactions, which likely involve syndecan-4, it attenuates mammary cancer cell migration independent of its metalloproteinase activity; however, its antiangiogenic action requires catalytic functionality, and its effects on metastasis in vivo are tissue niche-dependent.

Background & objectives. The ADAMTS proteinases are a family of secreted, matrix-associated enzymes that have diverse roles in the regulation of tissue organization and vascular homeostasis. Several of the 19 human family members have been identified as having either tumor promoting or suppressing roles. We previously demonstrated that decreased ADAMTS15 expression correlated with a worse clinical outcome in mammary carcinoma. Methods. We have explored the effects of A Disintegrin and Metalloproteinase with Thrombospondin motifs-15 (ADAMTS-15) on the behavior of MDA-MB-231 and MCF 7 breast cancer cells by stable expression of either a wild type (wt) or metalloproteinase-inactive (E362A) protein. Results. No effects on mammary cancer cell proliferation or apoptosis were observed for either form of ADAMTS-15. However, both forms reduced cell migration on fibronectin or laminin matrices, though motility on a Type I collagen matrix was unimpaired. Knockdown of syndecan-4 attenuated the inhibitory effects of ADAMTS-15 on cell migration. In contrast to its effects on cell migration, wt ADAMTS-15 but not the E362A inactive mutant inhibited endothelial tubulogenesis in 3D collagen gels and angiogenesis in the aortic ring assay. In experimental metastasis assays in nude mice, MDA-MB-231 cells expressing either form of ADAMTS-15 showed reduced spread to the liver, though lung colonization was enhanced for cells expressing wt ADAMTS-15. Conclusiona. These studies indicate that extracellular ADAMTS-15 has multiple actions on tumor pathophysiology. Via modulation of cell-ECM interactions, which likely involve syndecan-4, it attenuates mammary cancer cell migration independent of its metalloproteinase activity; however, its antiangiogenic action requires catalytic functionality, and its effects on metastasis in vivo are tissue niche-dependent.

The mammalian vascular system consists of two distinct, but closely related, networks: the blood vasculature (itself divided into arterial and venous networks) and the lymphatic vasculature. EC (endothelial cell) lineage specification has been proposed to be determined during embryonic development, after which the ECs are committed to their fate. However, increasing evidence suggests that ECs retain various degrees of plasticity, and have the ability to express characteristics of alternative cell lineages. Therapeutic control of endothelial plasticity will allow greater understanding of the genesis and treatment of several vascular diseases.