Tight junctions have recently emerged as essential signaling regulators of proliferation and differentiation in epithelial tissues. Here, we aimed to identify the factors regulating claudin-7 expression in the colon, and analyzed the consequences of claudin-7 overexpression in colorectal carcinoma (CRC). In healthy human colonic crypts, claudin-7 expression was found to be low in the stem/progenitor cell compartment, where Tcf-4 activity is high, but strong in differentiated and postmitotic cells, where Tcf-4 is inactive. In contrast, claudin-7 was overexpressed in areas with high Tcf-4 target gene levels in CRC samples. In vitro, Tcf-4 was able to repress claudin-7 expression, and the high mobility group-box transcription factor Sox-9 was identified as an essential mediator of this effect. Claudin-7 was strongly expressed in the intestine of Sox-9-deficient mice and in CRC cells with low Sox transcriptional activity. Sox-9 overexpression in these cells reinstated claudin-7 repression, and residual claudin-7 was no longer localized along the basolateral membrane, but was instead restricted to tight junctions. Using HT-29Cl.16E CRC cell spheroids, we found that Sox-9-induced polarization was completely reversed after virus-mediated claudin-7 overexpression. Claudin-7 overexpression in this context increased Tcf-4 target gene expression, proliferation, and tumorigenicity after injection in nude mice. Our results indicate that Tcf-4 maintains low levels of claudin-7 at the bottom of colonic crypts, acting via Sox-9. This negative regulation seems to be defective in CRC, possibly due to decreased Sox-9 activity, and the resulting claudin-7 overexpression promotes a loss of tumor cell polarization and contributes to tumorigenesis.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The alveolar subtype (ARMS) is clinically more aggressive, and characterized by an oncogenic fusion protein PAX3-FOXO1 that drives oncogenic cellular properties. Exosomes are small, secreted vesicles that affect paracrine signaling. We show that PAX3-FOXO1 transcript alters exosome content of C2C12 myoblasts, leading to pro-tumorigenic paracrine effects in recipient cells. Microarray analysis revealed alteration in miRNA content of exosomes, affecting cellular networks involved in cell metabolism, growth signaling, and cellular invasion. Overexpression and knockdown studies showed that miR-486-5p is an effector of PAX3-FOXO1, and mediates its paracrine effects in exosomes, including promoting recipient cell migration, invasion, and colony formation. Analysis of human RMS cells showed miR-486-5p is enriched in both cells and exosomes, and to a higher extent in ARMS subtypes. Analysis of human serum samples showed that miR-486-5p is enriched in exosomes of patients with RMS, and follow-up after chemotherapy showed decrease to control values. Our findings identify a novel role of both PAX3-FOXO1 and its downstream effector miR-486-5p in exosome-mediated oncogenic paracrine effects of RMS, and suggest its possible use as a biomarker.

Inherited and acquired changes in pre-mRNA processing have significant roles in human diseases, especially cancer. Characterization of aberrantly spliced mRNAs may thus contribute to understand malignant transformation. We recently reported an anti-oncogenic potential for the SOX9 transcription factor in the colon. For instance, the Sox9 gene knock out in the mouse intestine results in an excess of proliferation with appearance of hyperplasia. SOX9 is expressed in colon cancer cells but its endogenous activity is weak. We looked for SOX9 variants that may impair SOX9 activity in colon cancer cells and we discovered MiniSOX9, a truncated version of SOX9 devoid of transactivation domain as a result of retention of the second intron. A significant overexpression of MiniSOX9 mRNA in human tumor samples compared with their matched normal tissues was observed by realtime reverse transcriptase-PCR. Immunohistochemistry revealed that MiniSOX9 is expressed at high levels in human colon cancer samples whereas it is undetectable in the surrounding healthy tissues. Finally, we discovered that MiniSOX9 behaves as a SOX9 inhibitor, inhibits protein kinase Ca promoter activity and stimulates the canonical Wnt pathway. This potential oncogenic activity of the SOX9 locus gives new insights on its role in colon cancer.

A deletion of the transcription factor SOX9 gene in the mice intestine affects the morphology of the colon epithelium and leads to hyperplasia. Nevertheless, direct transcriptional targets of SOX9 in this tissue are still unknown. A microarray analysis identified the tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) as a possible SOX9 target gene and we demonstrate here that SOX9 upregulates CEA-CAM1 in human colonic cells. Moreover, CEACAM1 expression is reduced in colon of SOX9-deficient mouse, suggesting an important function for SOX9 in the transcriptional activation of the CEACAM1 gene. We further identified SOX9-binding sequences in the human and rat CEACAM1 promoters, and an electrophoretic mobility shift together with a chromatin immunoprecipitation provided an additional evidence of the SOX9 binding to the human promoter. In addition, we established that histone acyl-transferase p300 behaves as an SOX9 coactivator of the rat and human CEACAM1 promoters. These results highlight CEACAM1 as the first direct target of SOX9 identified in the colon epithelium.

Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in children. Despite multiple attempts at intensifying chemotherapeutic approaches to treatment, only moderate improvements in survival have been made for patients with advanced disease. Retinoic acid is a differentiation agent that has shown some antitumor efficacy in RMS cells in vitro; however, the effects are of low magnitude. E23-(4 0 -hydroxyl-3 0 -adamantylbiphenyl-4-yl) acrylic acid (ST1926) is a novel orally available synthetic atypical retinoid, shown to have more potent activity than retinoic acid in several types of cancer cells. We used in vitro and in vivo models of RMS to explore the efficacy of ST1926 as a possible therapeutic agent in this sarcoma. We found that ST1926 reduced RMS cell viability in all tested alveolar (ARMS) and embryonal (ERMS) RMS cell lines, at readily achievable micromolar concentrations in mice. ST1926 induced an early DNA damage response (DDR), which led to increase in apoptosis, in addition to S-phase cell cycle arrest and a reduction in protein levels of the cell cycle kinase CDK1. Effects were irrespective of TP53 mutational status. Interestingly, in ARMS cells, ST1926 treatment decreased PAX3-FOXO1 fusion oncoprotein levels, and this suppression occurred at a post-transcriptional level. In vivo, ST1926 was effective in inhibiting growth of ARMS and ERMS xenografts, and induced a prominent DDR. We conclude that ST1926 has preclinical efficacy against RMS, and should be further developed in this disease in clinical trials.Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma, and the third most common solid tumor in children. 1 It accounts for 6% of all childhood cancers and approximately 40% of soft tissue sarcomas. 2,3 RMS likely arises from primitive mesenchymal progenitors that have undergone a limited program of myogenic differentiation, because of the expression of skeletal myogenic proteins in RMS tumors. 1,4 Rhabdomyosarcoma occurs in two major histological subtypes: embryonal (ERMS) and alveolar (ARMS) histologies. 1,4 ARMS is associated with a chromosomal translocation between the PAX3 or PAX7 and FOXO1 genes in approximately 55 and 22% of cases, respectively. 5 Patients presenting with ARMS tumors usually have a worse prognosis as compared to ERMS. The PAX-FOXO1 fusion transcript has been shown to exhibit a more potent transcriptional activation function than the PAX proteins alone, 6 and contributes to the invasive phenotype of ARMS, 7,8 making it an interesting target for therapeutic intervention. 9 Despite multiple attempts at intensifying chemotherapeutic approaches to treatment, limited improvements in survival have been made for patients with advanced-stage disease or recurrent RMS over the past 10 years. [10][11][12] This underlies the need for novel therapeutic approaches. 10,13 Retinoic acid is a morphogen and a major regulator of cellular proliferation, apoptosis and differentiation, 14 and has been investigated as differentiation therapy in multiple types of cancer, contributi...

Rhabdomyosarcoma (RMS) is an aggressive childhood sarcoma with two distinct subtypes, embryonal (ERMS) and alveolar (ARMS) histologies. More effective treatment is needed to improve outcomes, beyond conventional cytotoxic chemotherapy. The pan-histone deacetylase inhibitor, Suberoylanilide Hydroxamic Acid (SAHA), has shown promising efficacy in limited preclinical studies. We used a panel of human ERMS and ARMS cell lines and xenografts to evaluate the effects of SAHA as a therapeutic agent in both RMS subtypes. SAHA decreased cell viability by inhibiting S-phase progression in all cell lines tested, and induced apoptosis in all but one cell line. Molecularly, SAHA-treated cells showed activation of a DNA damage response, induction of the cell cycle inhibitors p21 Cip1 and p27 Kip1 and downregulation of Cyclin D1. In a subset of RMS cell lines, SAHA promoted features of cellular senescence and myogenic differentiation. Interestingly, SAHA treatment profoundly decreased protein levels of the driver fusion oncoprotein PAX3-FOXO1 in ARMS cells at a post-translational level. In vivo, SAHA-treated xenografts showed increased histone acetylation and induction of a DNA damage response, along with variable upregulation of p21 Cip1 and p27 Kip1. However, while the ARMS Rh41 xenograft tumor growth was significantly inhibited, there was no significant inhibition of the ERMS tumor xenograft RD. Thus, our work shows that, while SAHA is effective against ERMS and ARMS tumor cells in vitro, it has divergent in vivo effects. Together with the observed effects on the PAX3-FOXO1 fusion protein, these data suggest SAHA as a possible therapeutic agent for clinical testing in patients with fusion protein-positive RMS.

The tumor suppressor p53, and the cyclin-dependent kinase inhibitor Ink4c, have been both implicated in spermatogenesis control. Both p53-/- and Ink4c-/- single knockout male mice are fertile, despite testicular hypertrophy, Leydig cell differentiation defect, and increased sperm count in Ink4c-/- males. To investigate their collaborative roles, we studied p53-/- Ink4c-/- dual knockout animals, and found that male p53-/- Ink4c-/- mice have profoundly reduced fertility. Dual knockout male mice show a marked decrease in sperm count, abnormal sperm morphology and motility, prolongation of spermatozoa proliferation and delay of meiosis entry, and accumulation of DNA damage. Genetic studies showed that the effects of p53 loss on fertility are independent of its downstream effector Cdkn1a. Absence of p53 also partially reverses the hyperplasia seen upon Ink4c loss, and normalizes the Leydig cell differentiation defect. These results implicate p53 in mitigating both the delayed entry into meiosis and the secondary apoptotic response that occur in the absence of Ink4c. We conclude that the cell cycle genes p53 and Ink4c collaborate in sperm cell development and differentiation, and may be important candidates to investigate in human male infertility conditions.

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