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PDF file, 25K, Characteristics of 51 breast cancer patients in the third cohort used to determine the clinical correlation of Slit2 mRNA expression (Fig. 6D).

PDF file, 44K, Robo2 knockdown did not promote the soft agar colony forming ability of MDA-MB-231 cells when co-cultured with fibroblasts.

PDF file, 25K, Characteristics of 162 breast cancer patients in the second cohort used to determine the clinical correlation of Robo1 protein expression (Fig. 6C).

Supplementary Figure 4 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

Supplementary Figure 1 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

PDF file, 60K, High expression of Robo1 in cancer cells or Slit2 in stromal fibroblasts prevents B-catenin nuclear translocation in breast cancer specimen.

Supplementary Figure 5 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

PDF file, 26K, Characteristics of 122 breast cancer patients in the first cohort used to determine the clinical correlation of Robo1 mRNA expression (Fig. 6A).

PDF file, 26K, Univariate and multivariate proportional hazards analysis of mortality of breast cancer patients of the second cohort.

PDF file, 34K, Activated Robo1 by rSlit2 interacted with p85 in BT20 cells.

PDF file, 73K, Robo1 protein expression in tumors derived from MDA-MB-231 and HCC1937 fat pad xenografts.

Supplementary Figure 3 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

Supplementary Figures 1-4 from Small Molecule Targeting the Hec1/Nek2 Mitotic Pathway Suppresses Tumor Cell Growth in Culture and in Animal

PDF file, 26K, Co-staining of Slit2 and (alpha)-SMA expression in fibroblasts of breast cancer specimen.

Supplementary Figure 2 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

PDF file, 23K, Comparable total B-catenin protein expressions in shCtrl and shRobo1 MDA-MB-231 cells.

The BRCA1-interacted transcriptional repressor ZBRK1 has been associated with antiangiogenesis, but direct evidence of a tumor suppressor role has been lacking. In this study, we provide evidence of such a role in cervical carcinoma. ZBRK1 levels in cervical tumor cells were significantly lower than in normal cervical epithelial cells. In HeLa cervical cancer cells, enforced expression inhibited malignant growth, invasion, and metastasis in a variety of in vitro and in vivo assays. Expression of the metalloproteinase MMP9, which is known to be an important driver of invasion and metastasis, was found to be inversely correlated with ZBRK1 in tumor tissues and a target for repression in tumor cells. Our findings suggest that ZBRK1 acts to inhibit metastasis of cervical carcinoma, perhaps by modulating MMP9 expression. Cancer Res; 70(1); 192–201

PDF file, 22K, List of primer sequences in the study.

Supplementary Figure Legends 1-5 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

Tumor microenvironment plays a critical role in regulating tumor progression by secreting factors that mediate cancer cell growth. Stromal fibroblasts can promote tumor growth through paracrine factors; however, restraint of malignant carcinoma progression by the microenvironment also has been observed. The mechanisms that underlie this paradox remain unknown. Here, we report that the tumorigenic potential of breast cancer cells is determined by an interaction between the Robo1 receptor and its ligand Slit2, which is secreted by stromal fibroblasts. The presence of an active Slit2/Robo1 signal blocks the translocation of β-catenin into nucleus, leading to downregulation of c-myc and cyclin D1 via the phosphoinositide 3-kinase (PI3K)/Akt pathway. Clinically, high Robo1 expression in the breast cancer cells correlates with increased survival in patients with breast cancer, and low Slit2 expression in the stromal fibroblasts is associated with lymph node metastasis. Together, our findings explain how a specific tumor microenvironment can restrain a given type of cancer cell from progression and show that both stromal fibroblasts and tumor cell heterogeneity affect breast cancer outcomes. Cancer Res; 72(18); 4652–61. ©2012 AACR.

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Supplementary Tables 1-2 from ZBRK1 Acts as a Metastatic Suppressor by Directly Regulating MMP9 in Cervical Cancer

ZBRK1, named after its structure of Zinc finger and BRCA1-interacting protein with KRAB domain-1 (ZBRK1), is a transcriptional repressor modulated by BRCA1. Recent evidence also indicated that ZBRK1 collaborated with BRCA1/CtIP to repress angiopoietin-1 expression in preventing over enlargement of blood vessels in tumors, suggesting that ZBRK1 may exert a critical role during tumor progression. However, a direct role of ZBRK1 in tumorigenesis and tumor progression remains obscure. Here we found that ZBRK1 expression was significantly lower in highly malignant cervical cancer cells than the counterpart normal tissue. Ectopic expression of ZBRK1 in HeLa cells significantly inhibits its neoplastic phenotypes including cell proliferation, soft-agar colony formation and tumor growth in nude mice. To explore its mechanisms, analyses of gene expression patterns of these cells revealed groups of genes not only critical for cell proliferation but also for cell motility being down regulated. Consistently, ectopic expression of ZBRK1 inhibits HeLa cells migration in cell migration and invasion assays in culture and metastatic assay in mice. Importantly, ZBRK1 directly represses transcription of the metastatic gene, MMP9, and the loss of ZBRK1 expression is inversely correlated to the elevated expression of MMP9 in cervical cancer specimens. Taken together, these results indicate that ZBRK1 may have a critical role as a tumor suppressor, especially in metastasis, through directly modulating metastatic genes such as MMP9.

Hec1 is a conserved mitotic regulator critical for spindle checkpoint control, kinetochore functionality, and cell survival. Overexpression of Hec1 has been detected in a variety of human cancers and is linked to poor prognosis of primary breast cancers. Through a chemical genetic screening, we have identified a small molecule, N-(4-[2,4-dimethyl-phenyl]-thiazol-2-yl)-benzamide (INH1), which specifically disrupts the Hec1/Nek2 interaction via direct Hec1 binding. Treating cells with INH1 triggered reduction of kinetochore-bound Hec1 as well as global Nek2 protein level, consequently leading to metaphase chromosome misalignment, spindle aberrancy, and eventual cell death. INH1 effectively inhibited the proliferation of multiple human breast cancer cell lines in culture (GI50, 10–21 μmol/L). Furthermore, treatment with INH1 retarded tumor growth in a nude mouse model bearing xenografts derived from the human breast cancer line MDA-MB-468, with no apparent side effects. This study suggests that the Hec1/Nek2 pathway may serve as a novel mitotic target for cancer intervention by small compounds. [Cancer Res 2008;68(20):8393–9]

ZBRK1, a zinc finger protein that interacts with breast cancer 1 (BRCA1) and KRAB-ZFP-associated protein 1 (KAP1), has been suggested to serve as a tumor suppressor via repression of tumor metastasis/invasion. To date, the detailed molecular mechanisms for how BRCA1 and KAP1 participate in ZBRK1-mediated transcriptional repression, metastasis and invasion as well as the associated clinical relevance remain unclear. In this study, we demonstrated that both the N- and C-terminal domains of ZBRK1 are important for inhibiting cell proliferation and anchorage-independent growth in cervical cancer. Specifically, the N-terminal KRAB domain of ZBRK1 displayed a more crucial role in inhibiting metastasis and invasion through modulation of KAP1 function in a transcriptionally dependent manner. The loss of ZBRK1 results in an increase of KAP1 expression, which enhanced migration and invasion of cervical cancer cells both the in vitro and in vivo. Moreover, an inverse correlation of expression levels was observed between ZBRK1 and KAP1 following tumor progression from in situ carcinoma to invasive/metastatic cervical cancer specimens. Taken together, the current results indicate that a loss of ZBRK1 contributes to the increased expression of KAP1, potentiating its role to enhance metastasis and invasion. Citation Format: Wei-Jan Wang, Li-Fang Lin, Chien-Feng Li, Wen-Ming Yang, Dennis Ding-Hwa Wang, Wen-Chang Chang, Wen-Hwa Lee, Ju-Ming Wang, Ju-Ming Wang. Loss of ZBRK1 contributes to the increase of KAP1 and promotes KAP1-mediated metastasis and invasion in cervical cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4926. doi:10.1158/1538-7445.AM2013-4926