The extracellular microenvironment is an integral component of normal and diseased tissues that is poorly understood owing to its complexity. To investigate the contribution of the microenvironment to lung fibrosis and adenocarcinoma progression, two pathologies characterized by excessive stromal expansion, we used mouse models to characterize the extracellular matrix (ECM) composition of normal lung, fibrotic lung, lung tumors, and metastases. Using quantitative proteomics, we identified and assayed the abundance of 113 ECM proteins, which revealed robust ECM protein signatures unique to fibrosis, primary tumors, or metastases. These analyses indicated significantly increased abundance of several S100 proteins, including Fibronectin and Tenascin-C (Tnc), in primary lung tumors and associated lymph node metastases compared with normal tissue. We further showed that Tnc expression is repressed by the transcription factor Nkx2-1, a well-established suppressor of metastatic progression. We found that increasing the levels of Tnc, via CRISPR-mediated transcriptional activation of the endogenous gene, enhanced the metastatic dissemination of lung adenocarcinoma cells. Interrogation of human cancer gene expression data revealed that high TNC expression correlates with worse prognosis for lung adenocarcinoma, and that a three-gene expression signature comprising TNC, S100A10, and S100A11 is a robust predictor of patient survival independent of age, sex, smoking history, and mutational load. Our findings suggest that the poorly understood ECM composition of the fibrotic and tumor microenvironment is an underexplored source of diagnostic markers and potential therapeutic targets for cancer patients.

Patterning of vertebrate melanophores is essential for mate selection and protection from UV-induced damage. Patterning can be influenced by circulating long-range factors, such as hormones, but it is unclear how their activity is controlled in recipient cells to prevent excesses in cell number and migration. The zebrafish wanderlust mutant harbors a mutation in the sheddase bace2 and exhibits hyperdendritic and hyperproliferative melanophores that localize to aberrant sites. We performed a chemical screen to identify suppressors of the wanderlust phenotype and found that inhibition of insulin/PI3Kγ/mTOR signaling rescues the defect. In normal physiology, Bace2 cleaves the insulin receptor, whereas its loss results in hyperactive insulin/PI3K/mTOR signaling. Insulin B, an isoform enriched in the head, drives the melanophore defect. These results suggest that insulin signaling is negatively regulated by melanophore-specific expression of a sheddase, highlighting how long-distance factors can be regulated in a cell-type-specific manner.

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Mounting evidence implicates the giant, cytoskeletal protein obscurin (720 to 870 kDa), encoded by the OBSCN gene, in the predisposition and development of breast cancer. Accordingly, prior work has shown that the sole loss of OBSCN from normal breast epithelial cells increases survival and chemoresistance, induces cytoskeletal alterations, enhances cell migration and invasion, and promotes metastasis in the presence of oncogenic KRAS. Consistent with these observations, analysis of Kaplan–Meier Plotter datasets reveals that low OBSCN levels correlate with significantly reduced overall and relapse-free survival in breast cancer patients. Despite the compelling evidence implicating OBSCN loss in breast tumorigenesis and progression, its regulation remains elusive, limiting any efforts to restore its expression, a major challenge given its molecular complexity and gigantic size (~170 kb). Herein, we show that OBSCN-Antisense RNA 1 ( OBSCN-AS1 ), a novel nuclear long-noncoding RNA (lncRNA) gene originating from the minus strand of OBSCN , and OBSCN display positively correlated expression and are downregulated in breast cancer biopsies. OBSCN-AS1 regulates OBSCN expression through chromatin remodeling involving H3 lysine 4 trimethylation enrichment, associated with open chromatin conformation, and RNA polymerase II recruitment. CRISPR-activation of OBSCN-AS1 in triple-negative breast cancer cells effectively and specifically restores OBSCN expression and markedly suppresses cell migration, invasion, and dissemination from three-dimensional spheroids in vitro and metastasis in vivo. Collectively, these results reveal the previously unknown regulation of OBSCN by an antisense lncRNA and the metastasis suppressor function of the OBSCN-AS1/OBSCN gene pair, which may be used as prognostic biomarkers and/or therapeutic targets for metastatic breast cancer.

The cell cycle progression controlled by cyclin-dependent kinases (CDKs) is counterbalanced by CDK inhibitors (CKIs). The CKIs have two families: CIP/KIP and INK. The CIP/KIP members are composed of p21WAF1/CIP1, p27KIP1 and p57KIP2, which bind to and inhibit both cyclin D-CDK4/6 kinases and cyclin-E/A-CDK2. The INK members consist of p15INK4B, p16INK4A, p18INK4C and p19INK4D. The members in this family only bind to and inhibit G1CDKs (CDK4 and CDK6). We have previously reported that p27KIP1 plays both a positive and a negative role in human myeloid leukemia cells. In this study, we investigated whether p18INK4C plays any role in TGF-β-mediated cell cycle control of human myeloid leukemia cells. TGF-β significantly inhibited proliferation of MV4-11 and TF-1 cells, with upregulation of p27KIP1and downregulation of multiple CDKs and cyclins. Surprisingly, TGF-β significantly inhibited the expression of p18INK4C, a traditional G1 CDK inhibitor. The inhibitory effect of TGFβ on p18INK4C was time dependent. The time course showed that early inhibition of p18INK4C occurred at 3h after initiating TGFβ treatment in the cells with a maximal inhibition of 70% being observed at 72h incubation with TGF-β. The control cells treated with the solvent for TGFβ did not show any significant changes in the levels of p18INK4C. As a sample loading control, the cells treated with or without TGF-β expressed about the same levels of actin.TGFβ-induced p18INK4C inhibition is also dose dependent with a maximal inhibition being detected by Western blot when 30ng/ml of TGFβ was added to the culture. Low concentrations (5ng/ml or less) did not markedly affect the expression of p18INK4C. The association of p18INK4C with CDK4 was also decreased in response to TGF-β treatment detected by immunoprecipitation. Our data also showed that the two cell lines tested barely expresses p15INK4B, p16INK4A, p19INK4D, and p21WAF1/CIP1 and these inhibitors do not appear to play an important role, either positively or negatively, in the cell cycle control of the leukemia cells. Our data suggest that p18INK4C may be required for cell cycle progression in human myeloid leukemia cells tested. Whether such a positive role of p18INK4C is caused by a possible gene mutation(s) and thereby contributes to leoplasia of the leukemia cells is currently under investigation (supported by NIH-NIGMS MBRS RISE: R25 GM059244, Barry University). . Citation Format: Talia Guardia, Reshma Badaloo, Valentina Serrano, Alejandra Toro, Xiaotang Hu. Transforming growth factor-beta (TGF-β) inhibits growth of human myeloid leukemia cells and downregulates the expression of CDK inhibitor, p18INK4C. [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 576. doi:10.1158/1538-7445.AM2013-576

The cell cycle progression is controlled by cyclin-dependent kinases (CDKs). CDK-Activating Kinase (CAK) is an enzyme complex that is capable of phosphorylating (activating) all CDKs and is essential for G1 and G2 CDK activities. CAK is composed of CDK7, cyclin H, and Mat 1. CDK (CDK1 and CDK2) activity is also controlled by two inhibitory phosphorylation sites (tyrosine 15 and threonine 14) on the CDK. Dephosphorylation of these two sites by cdc25c activates CDK activity. We have previously reported that TGFβ inhibits growth of human myeloid leukemia cells via downregulation of multiple CDKs and cyclins. In this study, we investigated the effect of TGFβ on activity and expression of CAK and cdc25, the two upstream molecules of cell cycle regulatory molecules in human myeloid leukemia cells. TGF-β inhibited the proliferation of TF-1 and MV4-11 accompanied by an increase in p27 level and decrease in CDK1, CDK2, CDK4, cyclin A, cyclin D3, and cylcin B. However, TGF-β had no effect on the expression of CDK7, CDK 9, CDK11, cyclin H, Mat1, and their complexes (CDK7-cyclin H and CDK7-Mat1) for the time period tested up to 72 hours, detected by Western blot and immunoprecipitation, respectively. There were no detectable changes in the phosphorylation status of CDK7, CDK9, CDK11 in the cells treated with TGF-β as compared with the cells without TGFβ. In contrast, these cells showed marked decrease in the levels of MAT1 and CDK7 in response to retinoic acid and phorbol ester (PMA) stimulation. On the other hand, TGFβ significantly downregulated expression of cdc25c starting from 3h and with a maximum inhibition being observed at 72h. We also observed a transient dephosphorylation (activation) of cdc25c followed by significant decrease in the amount of dephophorylated 25c at 48 and 72h. Taken together, our data suggest that TGFβ-induced growth inhibition of human myeloid leukemia cells is CAK-independent but is linked to inhibition of early entry into mitosis of the cells by downregulating cdc25c-CDK1 pathway. Most likely, the downregulation of multiple CDKs and cyclins by TGFβ is regulated at translational or posttranslational but not transcriptional levels, because TGFβ had no any effect on the expression and phosphorylation of the CDKs that are involved in regulation of transcription factor TFII (CDK7 and CDK11) and TAK/P-TEFb (CDK9), respectively. (Supported by Faculty Incentive Grant and NIH-NIGMS MBRS RISE: R25 GM059244-13, Barry University). Citation Format: Alejandra Toro, Daria Vasilyeva, Talia Guardia, Tamara Guardia, Jazmine Duran, Xiaotang Hu. Transforming growth factor-beta (TGFβ)-mediated growth inhibition of human myeloid leukemia cells is CAK-independent but links to cdc25c-CDK1 pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3470. doi:10.1158/1538-7445.AM2014-3470