. relation and complete linkage as the distance metric and clustering method, respectively. The output data were loaded into Java Tree View software to generate heatmaps (66).Statistics. Statistical differences between different experimental groups were determined by Student's t test (2 tailed). The reported values represent the mean ± SD as indicated in the figures. A P value less than 0.05 was considered statistically significant. No samples were excluded from the analysis.Study approval. All animal experimental procedures were approved by the University of Florida IACUC.

Epigenetic alteration is a hallmark of all cancers. Such alterations lead to modulation of fundamental cancer-related functions, such as proliferation, migration, and invasion. In particular, methylation of Histone H3 Lysine 4 (H3K4), a histone mark generally associated with transcriptional activation, is altered during progression of several human cancers. While the depletion of H3K4 demethylases promotes breast cancer metastasis, the effect of H3K4 methyltransferases on metastasis is not clear. Nevertheless, gene duplications in the human SETD1A (hSETD1A) H3K4 methyltransferase are present in almost half of breast cancers. Herein, expression analysis determined that hSETD1A is upregulated in multiple metastatic human breast cancer cell lines and clinical tumor specimens. Ablation of hSETD1A in breast cancer cells led to a decrease in migration and invasion in vitro and to a decrease in metastasis in nude mice. Furthermore, a group of matrix metalloproteinases (including MMP2, MMP9, MMP12, MMP13, and MMP17) were identified which were downregulated upon depletion of hSETD1A and demonstrated a decrease in H3K4me3 at their proximal promoters based on chromatin immunoprecipitation analysis. These results provide evidence for a functional and mechanistic link among hSETD1A, MMPs, and metastasis in breast cancer, thereby supporting an oncogenic role for hSETD1A in cancer.Implications: This study reveals that hSETD1A controls tumor metastasis by activating MMP expression and provides an epigenetic link among hSETD1A, MMPs, and metastasis of breast cancer.

Key Points• ZNF24 represses VEGF transcription through direct binding to an 11-bp fragment of the VEGF proximal promoter.• ZNF24 functions as a negative regulator of developmental and tumor angiogenesis by inhibiting VEGF transcription.VEGF is a key regulator of normal and pathologic angiogenesis. Although many trans-activating factors of VEGF have been described, the transcriptional repression of VEGF remains much less understood. We have previously reported the identification of a SCAN domain-containing C2H2 zinc finger protein, ZNF24, that represses the transcription of VEGF. In the present study, we identify the mechanism by which ZNF24 represses VEGF transcription. Using reporter gene and electrophoretic mobility shift assays, we identify an 11-bp fragment of the proximal VEGF promoter as the ZNF24-binding site that is essential for ZNF24-mediated repression. We demonstrate in 2 in vivo models the potent inhibitory effect of ZNF24 on the vasculature. Expression of human ZNF24 induced in vivo vascular defects consistent with those induced by VEGF knockdown using a transgenic zebrafish model. These defects could be rescued by VEGF overexpression. Overexpression of ZNF24 in human breast cancer cells also inhibited tumor angiogenesis in an in vivo tumor model. Analyses of human breast cancer tissues showed that ZNF24 and VEGF levels were inversely correlated in malignant compared with normal tissues. These data demonstrate that ZNF24 represses VEGF transcription through direct binding to an 11-bp fragment of the VEGF proximal promoter and that it functions as a negative IntroductionVEGF is a key angiogenic factor during embryogenesis, as demonstrated by the embryonic lethality and abnormal blood vessel development of mice lacking a single VEGF allele. 1 It also plays critical roles in normal physiologic processes such as maintaining vascular stability 2 and promoting skeletal muscle differentiation. 3 Across a wide range of human cancers, VEGF serves as a principle proangiogenic factor. For example, VEGF levels are elevated in breast cancer patients 4 and are inversely correlated with patient survival. 5 Transcriptional regulation of VEGF has been defined in part by its many transcriptional activators, including Sp1, HIF-1, and STAT3. 6,7 In contrast, only the p53 family proteins and the von Hippel-Lindau (VHL) tumor suppressor have been discovered and well characterized as transcriptional repressors of VEGF.Zinc finger protein 24 (ZNF24, also known as ZNF191 or Kox17) was originally identified in an attempt to discover novel C2H2 zinc finger proteins in the hematopoietic system. 8 It possesses 4 Kruppel-like C2H2 zinc finger domains within the c-terminus that function as DNA-binding domains. 9 These domains bind to the TCAT repeats of the HUMTH01 microsatellite located in the first intron of the tyrosine hydroxylase gene in in vitro studies. 9,10 No in vivo function relevant to this observation has been documented to date. The N-terminus of ZNF24 contains a SCAN box domain that primarily serves as a dimerizat...

It is estimated that approximately 90% of patients with advanced prostate cancer develop bone metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of prognosis. The development of novel therapeutic strategies that impair the metastatic process and associated skeletal adversities is therefore critical to improving prostate cancer patient survival. Recognition of the importance of Cathepsin L (CTSL) to metastatic dissemination of cancer cells has led to the development of several CTSL inhibition strategies. The present investigation employed intra-cardiac injection of human PC-3ML prostate cancer cells into nude mice to examine tumor cell dissemination in a preclinical bone metastasis model. CTSL knockdown confirmed the validity of targeting this protease and subsequent intervention studies with the small molecule CTSL inhibitor KGP94 resulted in a significant reduction in metastatic tumor burden in the bone and an improvement in overall survival. CTSL inhibition by KGP94 also led to a significant impairment of tumor initiated angiogenesis. Furthermore, KGP94 treatment decreased osteoclast formation and bone resorptive function, thus, perturbing the reciprocal interactions between tumor cells and osteoclasts within the bone microenvironment which typically result in bone loss and aggressive growth of metastases. These functional effects were accompanied by a significant downregulation of NFκB signaling activity and expression of osteoclastogenesis related NFκB target genes. Collectively, these data indicate that the CTSL inhibitor KGP94 has the potential to alleviate metastatic disease progression and associated skeletal morbidities and hence may have utility in the treatment of advanced prostate cancer patients.