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MOV file 2346K, Video showing dynamic flow adhesion assays of ZR75.1 shNTC and shST6 cells on a HUVEC monolayer

PDF file 149K, In vivo validation of 3 hits identified in the screen (Mre11a, Fen1, Wwc1)

PDF file 174K, Additional characterization of the 4T1-Luc cells in lung retention assays

PDF file 95K, Data demonstrating that siRNA mediated downregulation of ST6GalNAc2 expression in human and mouse cell lines promotes tumor cell aggregation in vivo

MOV file 2886K, Video showing dynamic flow adhesion assays of ZR75.1 shNTC and shST6 cells on a HUVEC monolayer in the presence of the galectin-3 inhibitor GCS-100

PDF file 299K, Additional characterization of human breast cancer cell lines; immunofluorescence analysis and lung retention assays

PDF file 60K, Characterization of the MDA-MB-231 cells stably expressing vector or mouse ST6GalNAc2

PDF file 107K, Inventory of supplementary material. Supplementary Table S1 listing primers and siRNAs. Legends for Supplementary Fig. S1 - S7 Legends for Supplementary Movies S1 and S2 Supplementary Figures S1 - S6 legends Supplementary Movies S1 and S2 legends

Supplementary Table 1, Figures 1-5 from The Collagen Receptor Endo180 (CD280) Is Expressed on Basal-like Breast Tumor Cells and Promotes Tumor Growth In vivo

Tumor cell invasion into the surrounding stroma requires increased cell motility and extensive remodeling of the extracellular matrix. Endo180 (CD280, MRC2, urokinase-type plasminogen activator receptor-associated protein) is a recycling endocytic receptor that functions in both these cellular activities by promoting cell migration and uptake of collagens for intracellular degradation. In the normal breast, Endo180 is predominantly expressed by stromal fibroblasts. The contrary observation that Endo180 is expressed on epithelial tumor cell lines that display a high invasive capacity suggested that up-regulation of this receptor may be an associated and functional component in the acquisition of a more aggressive phenotype by tumor cells in vivo. Here, we show that high levels of Endo180 are found in a subset of basal-like breast cancers and that this expression is an independent prognostic marker for shorter disease-free survival. Two potential mechanisms for Endo180 up-regulation were uncovered. First, it was shown that Endo180 can be transcriptionally up-regulated in vitro following transforming growth factor-β treatment of breast cancer cells. Second, a proportion of Endo180+ tumors were shown to have Endo180 gene copy number gains and amplifications. To investigate the functional consequence of Endo180 up-regulation, MCF7 cells transfected with Endo180 were inoculated into immunocompromised mice. Expression of wild-type Endo180, but not an internalization-defective Endo180 mutant, resulted in enhanced tumor growth together with a reduction in tumor collagen content. Together, these data argue that elevated expression of this receptor in tumor cells could have important consequences in subsets of basal-like carcinomas for which there is a current lack of effective treatment. [Cancer Res 2007;67(21):10230–11]

To interrogate the complex mechanisms involved in the later stages of cancer metastasis, we designed a functional in vivo RNA interference (RNAi) screen combined with next-generation sequencing. Using this approach, we identified the sialyltransferase ST6GalNAc2 as a novel breast cancer metastasis suppressor. Mechanistically, ST6GalNAc2 silencing alters the profile of O-glycans on the tumor cell surface, facilitating binding of the soluble lectin galectin-3. This then enhances tumor cell retention and emboli formation at metastatic sites leading to increased metastatic burden, events that can be completely blocked by galectin-3 inhibition. Critically, elevated ST6GALNAC2, but not galectin-3, expression in estrogen receptor–negative breast cancers significantly correlates with reduced frequency of metastatic events and improved survival. These data demonstrate that the prometastatic role of galectin-3 is regulated by its ability to bind to the tumor cell surface and highlight the potential of monitoring ST6GalNAc2 expression to stratify patients with breast cancer for treatment with galectin-3 inhibitors. Significance: RNAi screens have the potential to uncover novel mechanisms in metastasis but do not necessarily identify clinically relevant therapeutic targets. Our demonstration that the sialyltransferase ST6GalNAc2 acts as a metastasis suppressor by impairing binding of galectin-3 to the tumor cell surface offers the opportunity to identify patients with breast cancer suitable for treatment with clinically well-tolerated galectin-3 inhibitors. Cancer Discov; 4(3); 304–17. ©2014 AACR. See related commentary by Ferrer and Reginato, p. 275 This article is highlighted in the In This Issue feature, p. 259

Purpose: The oncogenic drivers of triple-negative (TN) and basal-like breast cancers are largely unknown. Substantial evidence now links aberrant signaling by the fibroblast growth factor receptors (FGFR) to the development of multiple cancer types. Here, we examined the role of FGFR signaling in TN breast cancer. Experimental Design: We examined the sensitivity of a panel of 31 breast cancer cell lines to the selective FGFR inhibitor PD173074 and investigated the potential mechanisms underlying sensitivity. Results: TN breast cancer cell lines were more sensitive to PD173074 than comparator cell lines (P = 0.011), with 47% (7/15) of TN cell lines showing significantly reduced growth. The majority of TN cell lines showed only modest sensitivity to FGFR inhibition in two-dimensional growth but were highly sensitive in anchorage-independent conditions. PD173074 inhibited downstream mitogen-activated protein kinase and PI3K–AKT signaling and induced cell-cycle arrest and apoptosis. Basal-like breast cancer cell lines were found to express FGF2 ligand (11/21 positive) and, similarly, 62% of basal-like breast cancers expressed FGF2, as assessed by immunohistochemistry compared with 5% of nonbasal breast cancers (P < 0.0001). RNA interference targeting of FGF2 in basal-like cell lines significantly reduced growth in vitro and reduced down stream signaling, suggesting an autocrine FGF2 signaling loop. Treatment with PD173074 significantly reduced the growth of CAL51 basal-like breast cancer cell line xenografts in vivo. Conclusions: Basal-like breast cancer cell lines, and breast cancers, express autocrine FGF2 and show sensitivity to FGFR inhibitors, identifying a potential novel therapeutic approach for these cancers. Clin Cancer Res; 17(16); 5275–86. ©2011 AACR.

Tumor cell invasion into the surrounding stroma requires increased cell motility and extensive remodeling of the extracellular matrix. Endo180 (CD280, MRC2, urokinase-type plasminogen activator receptor-associated protein) is a recycling endocytic receptor that functions in both these cellular activities by promoting cell migration and uptake of collagens for intracellular degradation. In the normal breast, Endo180 is predominantly expressed by stromal fibroblasts. The contrary observation that Endo180 is expressed on epithelial tumor cell lines that display a high invasive capacity suggested that up-regulation of this receptor may be an associated and functional component in the acquisition of a more aggressive phenotype by tumor cells in vivo. Here, we show that high levels of Endo180 are found in a subset of basal-like breast cancers and that this expression is an independent prognostic marker for shorter disease-free survival. Two potential mechanisms for Endo180 up-regulation were uncovered. First, it was shown that Endo180 can be transcriptionally up-regulated in vitro following transforming growth factor-β treatment of breast cancer cells. Second, a proportion of Endo180+ tumors were shown to have Endo180 gene copy number gains and amplifications. To investigate the functional consequence of Endo180 up-regulation, MCF7 cells transfected with Endo180 were inoculated into immunocompromised mice. Expression of wild-type Endo180, but not an internalization-defective Endo180 mutant, resulted in enhanced tumor growth together with a reduction in tumor collagen content. Together, these data argue that elevated expression of this receptor in tumor cells could have important consequences in subsets of basal-like carcinomas for which there is a current lack of effective treatment. [Cancer Res 2007;67(21):10230–11]

BRCA1 and BRCA2 are important for DNA double-strand break repair by homologous recombination, and mutations in these genes predispose to breast and other cancers. Poly(ADP-ribose) polymerase (PARP) is an enzyme involved in base excision repair, a key pathway in the repair of DNA single-strand breaks. We show here that BRCA1 or BRCA2 dysfunction unexpectedly and profoundly sensitizes cells to the inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis. This seems to be because the inhibition of PARP leads to the persistence of DNA lesions normally repaired by homologous recombination. These results illustrate how different pathways cooperate to repair damage, and suggest that the targeted inhibition of particular DNA repair pathways may allow the design of specific and less toxic therapies for cancer.