Ovarian Tumor Cell Expression of Claudin-4 Reduces Apoptotic Response to Paclitaxel.

A significant factor contributing to poor survival rates for patients with ovarian cancer is the insensitivity of tumors to standard-of-care chemotherapy. In this study, we investigated the effect of claudin-4 expression on ovarian tumor cell apoptotic response to cisplatin and paclitaxel. We manipulated claudin-4 gene expression by silencing expression [short hairpin RNA (shRNA)] in cells with endogenously expressed claudin-4 or overexpressing claudin-4 in cells that natively do not express claudin-4. In addition, we inhibited claudin-4 activity with a claudin mimic peptide (CMP). We monitored apoptotic response by caspase-3 and Annexin V binding. We examined proliferation rate by counting the cell number over time as well as measuring the number of mitotic cells. Proximity ligation assays, immunoprecipitation (IP), and immunofluorescence were performed to examine interactions of claudin-4. Western blot analysis of tubulin in cell fractions was used to determine the changes in tubulin polymerization with changes in claudin-4 expression. Results show that claudin-4 expression reduced epithelial ovarian cancer (EOC) cell apoptotic response to paclitaxel. EOCs without claudin-4 proliferated more slowly with enhanced mitotic arrest compared with the cells expressing claudin-4. Furthermore, our results indicate that claudin-4 interacts with tubulin, having a profound effect on the structure and polymerization of the microtubule network. In conclusion, we demonstrate that claudin-4 reduces the ovarian tumor cell response to microtubule-targeting paclitaxel and disrupting claudin-4 with CMP can restore apoptotic response. IMPLICATIONS: These results suggest that claudin-4 expression may provide a biomarker for paclitaxel response and can be a target for new therapeutic strategies to improve response.

A tRNA fragment, tRF5-Glu, regulates BCAR3 expression and proliferation in ovarian cancer cells.

Ovarian cancer is a complex disease marked by tumor heterogeneity, which contributes to difficulties in diagnosis and treatment. New molecular targets and better molecular profiles defining subsets of patients are needed. tRNA fragments (tRFs) offer a recently identified group of noncoding RNAs that are often as abundant as microRNAs in cancer cells. Initially their presence in deep sequencing data sets was attributed to the breakdown of mature tRNAs, however, it is now clear that they are actively generated and function in multiple regulatory events. One such tRF, a 5' fragment of tRNA-Glu-CTC (tRF5-Glu), is processed from the mature tRNA-Glu and is shown in this study to be expressed in ovarian cancer cells. We confirmed that tRF5-Glu binds directly to a site in the 3'UTR of the Breast Cancer Anti-Estrogen Resistance 3 (BCAR3) mRNA thereby down regulating its expression. BCAR3 has not previously been studied in ovarian cancer cells and our studies demonstrate that inhibiting BCAR3 expression suppresses ovarian cancer cell proliferation. Furthermore, mimics of tRF5-Glu were found to inhibit proliferation of ovarian cancer cells. In summary, BCAR3 and tRF5-Glu contribute to the complex tumor heterogeneity of ovarian cancer cells and may provide new targets for therapeutic intervention.

Claudin-4 activity in ovarian tumor cell apoptosis resistance and migration.

BACKGROUND: Claudin-4 is a transmembrane protein expressed at high levels in the majority of epithelial ovarian tumors, irrespective of subtype, and has been associated with tumor cells that are both chemoresistant and highly mobile. The objective of this study was to determine the functional role that claudin-4 plays in apoptosis resistance and migration as well as the therapeutic utility of targeting claudin-4 activity with a small mimic peptide. METHODS: We examined claudin-4 activity in human ovarian tumor cell lines (SKOV3, OVCAR3, PEO4) using in vitro caspase and scratch assays as well as an in vivo mouse model of ovarian cancer. Claudin-4 activity was disrupted by treating cells with a small peptide that mimics the DFYNP sequence in the second extracellular loop of claudin-4. Claudin-4 expression was also altered using shRNA-mediated gene silencing. RESULTS: Both the disruption of claudin-4 activity and the loss of claudin-4 expression significantly increased tumor cell caspase-3 activation (4 to 10-fold, respectively) in response to the apoptotic inducer staurosporine and reduced tumor cell migration by 50 %. The mimic peptide had no effect on cells that lacked claudin-4 expression. Female athymic nude mice bearing ZsGreen-PEO4 ovarian tumors showed a significant decrease in ovarian tumor burden, due to increased apoptosis, after treatment with intraperitoneal injections of 4 mg/kg mimic peptide every 48 h for three weeks, compared to control peptide treated mice. CONCLUSION: Claudin-4 functionally contributes to both ovarian tumor cell apoptosis resistance and migration and targeting extracellular loop interactions of claudin-4 may have therapeutic implications for reducing ovarian tumor burden.

Roles for ß-catenin and doxycycline in the regulation of respiratory epithelial cell frequency and function.

The expression of ß-catenin-dependent genes can be increased through the Cre recombinase (Cre)-mediated elimination of the exon 3-encoded sequence. This mutant ß-catenin is termed DE3, and promotes the expression of ß-catenin-dependent genes. Our previous study used the DE3 model to demonstrate that persistent ß-catenin activity inhibited bronchiolar Clara-to-ciliated cell differentiation. The present study was designed to evaluate the roles of ß-catenin in regulating the tracheal progenitor cell hierarchy. However, initial experiments demonstrated that the tetracycline-responsive element-Cre transgene (TRE-Cre) was active in the absence of a reverse tetracycline transactivator driver or inducer, doxycycline (Dox). This spurious TRE-Cre transgene activity was not detected using the ROSA26-floxed STOP-LacZ reporter. To determine if the phenotype was a consequence of genotype or treatment with Dox, tracheal and lung specimens were evaluated using quantitative histomorphometric techniques. Analyses of uninduced mice demonstrated a significant effect of genotype on tracheal epithelial cell mass, involving basal, Clara-like cell types. The bronchial and bronchiolar Clara cell mass was also decreased. Paradoxically, an effect on ciliated cell mass was not detected. Activation of the ß-catenin reporter transgene TOPGal demonstrated that ß-catenin-dependent gene expression led to the genotype-dependent tracheal and bronchiolar phenotype. Comparative analyses of wild-type or keratin 14-rtTA(+/0)/TRE-cre(+/0)/DE3(+/+) mice receiving standard or Dox chow demonstrated an effect of treatment with Dox on basal, Clara-like, and Clara cell masses. We discuss these results in terms of cautionary notes and with regard to alterations of progenitor cell hierarchies in response to low-level injury.

Context-dependent differentiation of multipotential keratin 14-expressing tracheal basal cells.

Multipotential (MP) differentiation is one characteristic of a tissue-specific stem cell (TSC). Lineage tracing of tracheobronchial basal cells after naphthalene (NA) injury or in the postnatal period demonstrated that basal cells were MP progenitors for Clara-like and ciliated cells. These studies, as well as reports of spatially restricted, label-retaining basal cells, and MP differentiation by human bronchial cells support the hypothesis that a TSC maintained and repaired the tracheobronchial epithelium. However, differences in basal cell phenotype (keratin [K] 5+ versus K14+), age (postnatal versus adult), health status (normal versus injured), and injury type (acid, detergent, NA) limited comparisons among studies and thus diminished the strength of the TSC argument. The finding that K14 was up-regulated after NA injury was a caveat to our previous analysis of reparative (r)K14-expressing cells (EC). Thus, the present study lineage traced steady-state (s)K14EC and evaluated differentiation potential in the normal and repairing epithelium. We showed that sK14EC were unipotential in the normal epithelium and MP after NA, sK14EC-dervied clones were not restricted to putative TSC niches, sK14EC cells were a direct progenitor for Clara-like and ciliated cells, MP-sK14EC clones accumulated over time, and sK14EC-derived Clara-like cells were progenitors for ciliated cells.