CASC4/GOLM2 drives high grade serous carcinoma anoikis resistance through the recycling of EGFR.

Ovarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian carcinoma (HGSC) subtype accounts for almost 70% of ovarian cancers and is the deadliest. HGSC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. HGSC survival in peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). Most anoikis resistant mechanisms are dependent on microenvironment interactions with cell surface-associated proteins, such as integrins and receptor tyrosine kinases (RTKs). We previously identified the gene CASC4 as a driver of anoikis resistance. CASC4 is predicted to be a Golgi-associated protein that may regulate protein trafficking to the plasma membrane, but CASC4 is largely uncharacterized in literature; thus, we sought to determine how CASC4 confers anoikis resistance to HGSC cells. Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we cultured three human HGSC cell lines (PEO1, CaOV3, OVCAR3), and a murine HGSC cell line, (ID8) with shRNA-mediated CASC4 knockdowns (CASC4 KD) in suspension, to recapitulate the peritoneal fluid environment in vitro. CASC4 KD significantly inhibited cell proliferation and colony formation ability, and increased apoptosis. A Reverse Phase Protein Assay (RPPA) showed that CASC4 KD resulted in a broad re-programming of membrane-associated proteins. Specifically, CASC4 KD led to decreased protein levels of the RTK Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways, leading us to hypothesize that CASC4 drives HGSC survival through mediating recycling and trafficking of EGFR. Indeed, loss of CASC4 led to a decrease in both EGFR membrane localization, reduced turnover of EGFR, and increased EGFR ubiquitination. Moreover, a syngeneic ID8 murine model of ovarian cancer showed that knocking down CASC4 leads to decreased tumor burden and dissemination.

Bone Structure and Turnover in Postmenopausal Women With Long-Standing Type 1 Diabetes.

Compromised bone structural and mechanical properties are implicated in the increased fracture risk in type 1 diabetes (T1D). We investigated bone structure and turnover by histomorphometry in postmenopausal women with T1D and controls without diabetes using tetracycline double-labeled transiliac bone biopsy. After in vivo tetracycline double labeling, postmenopausal women with T1D of at least 10 years and without diabetes underwent transiliac bone biopsy. An expert blinded to the study group performed histomorphometry. Static and dynamic histomorphometry measurements were performed and compared between the two groups. The analysis included 9 postmenopausal women with T1D (mean age 58.4 ± 7.1 years with 37.9 ± 10.9 years of diabetes and HbA1c 7.1% ± 0.4%) and 7 postmenopausal women without diabetes (mean age 60.9 ± 3.3 years and HbA1c 5.4% ± 0.2%). There were no significant differences in serum PTH (38.6 ± 8.1 versus 51.9 ± 23.9 pg/mL), CTX (0.4 ± 0.2 versus 0.51 ± 0.34 ng/mL), or P1NP (64.5 ± 26.2 versus 87.3 ± 45.3 ng/mL). Serum 25-hydroxyvitamin D levels were higher in T1D than in controls (53.1 ± 20.8 versus 30.9 ± 8.2 ng/mL, p < 0.05). Bone structure metrics (bone volume, trabecular thickness, trabecular number, and cortical thickness) were similar between the groups. Indices of bone formation (osteoid volume, osteoid surface, and bone formation rate) were 40% lower in T1D and associated with lower activation frequency. However, the differences in bone formation were not statistically significant. Long-standing T1D may affect bone turnover, mainly bone formation, without significantly affecting bone structure. Further research is needed to understand bone turnover and factors affecting bone turnover in people with T1D. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

ATF6-Mediated Signaling Contributes to PARP Inhibitor Resistance in Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is the deadliest ovarian cancer histotype due in-part to the lack of therapeutic options for chemotherapy-resistant disease. PARP inhibitors (PARPi) represent a targeted treatment. However, PARPi resistance is becoming a significant clinical challenge. There is an urgent need to overcome resistance mechanisms to extend disease-free intervals. We established isogeneic PARPi-sensitive and -resistant HGSOC cell lines. In three PARPi-resistant models, there is a significant increase in AP-1 transcriptional activity and DNA repair capacity. Using RNA-sequencing and an shRNA screen, we identified activating transcription factor 6 (ATF6) as a mediator of AP-1 activity, DNA damage response, and PARPi resistance. In publicly available datasets, ATF6 expression is elevated in HGSOC and portends a poorer recurrence-free survival. In a cohort of primary HGSOC tumors, higher ATF6 expression significantly correlated to PARPi resistance. In PARPi-resistant cell lines and a PDX model, inhibition of a known ATF6 regulator, p38, attenuated AP-1 activity and RAD51 foci formation, enhanced DNA damage, significantly inhibited tumor burden, and reduced accumulation of nuclear ATF6. IMPLICATIONS: This study highlights that a novel p38-ATF6-mediated AP-1 signaling axis contributes to PARPi resistance and provides a clinical rationale for combining PARPi and AP-1 signaling inhibitors.

Inhibiting Wnt/beta-catenin in CTNNB1-mutated endometrial cancer.

The role of ß-catenin/TCF transcriptional activity in endometrial cancer (EC) recurrence is not well understood. We assessed the impact of Wnt/ß-catenin inhibition in EC models. In an analysis of the Cancer Genome Atlas, we confirmed that CTNNB1 mutations are enriched in recurrent low-risk EC and showed that aberrant Wnt/ß-catenin pathway activation is associated with recurrence. We studied CTNNB1-wildtype (HEC1B, Ishikawa) and CTNNB1-mutant (HEC108, HEC265, HEC1B-S33Y, Ishikawa-S33Y) EC cell lines. Dose response curves were determined for 5 Wnt/ß-catenin pathway inhibitors (Wnt-C59, XAV-939, PyrPam, PRI-724, SM04690). XAV939, Wnt-C59 and PyrPam inhibited function upstream of ß-catenin transcriptional activity and were ineffective at inhibiting cell viability. In contrast, PRI724 and SM04690 indirectly inhibited ß-catenin transcriptional activity and significantly reduced cell viability in CTNNB1-mutant cell lines. Treatment with SM04690 reduced cell viability (Licor Cell stain) in all EC cell lines, but viability was significantly lower in CTNNB1-mutant cell lines (p < 0.01). Mechanistically, SM04690 significantly inhibited proliferation measured via 5'-bromo-2'-deoxyuridine incorporation and reduced T cell factor (TCF) transcriptional activity. HEC1B, HEC1B-S33Y and HEC265 tumor-bearing mice were treated with vehicle or SM04690. Tumors treated with SM04690 had smaller mean volumes than those treated with vehicle (p < 0.001, p = 0.014, p = 0.06). In HEC1B-S33Y and HEC265 tumors, SM04690 treatment significantly reduced Ki67 H-scores compared to vehicle (p = 0.035, p = 0.024). Targeting the Wnt/ß-catenin pathway in CTNNB1-mutant EC effectively inhibited proliferation and ß-catenin/TCF transcriptional activity and blunted tumor progression in in vivo models. These studies suggest ß-catenin transcriptional inhibitors are effective in EC and particularly in CTNNB1-mutant EC, highlighting a potential therapeutic vulnerability for treatment of CTNNB1-mutant EC.

Targeting Fatty Acid Oxidation to Promote Anoikis and Inhibit Ovarian Cancer Progression.

Epithelial-derived high-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy. Roughly 80% of patients are diagnosed with late-stage disease, which is defined by wide-spread cancer dissemination throughout the pelvic and peritoneal cavities. HGSOC dissemination is dependent on tumor cells acquiring the ability to resist anoikis (apoptosis triggered by cell detachment). Epithelial cell detachment from the underlying basement membrane or extracellular matrix leads to cellular stress, including nutrient deprivation. In this report, we examined the contribution of fatty acid oxidation (FAO) in supporting anoikis resistance. We examined expression Carnitine Palmitoyltransferase 1A (CPT1A) in a panel of HGSOC cell lines cultured in adherent and suspension conditions. With CPT1A knockdown cells, we evaluated anoikis by caspase 3/7 activity, cleaved caspase 3 immunofluorescence, flow cytometry, and colony formation. We assessed CPT1A-dependent mitochondrial activity and tested the effect of exogenous oleic acid on anoikis and mitochondrial activity. In a patient-derived xenograft model, we administered etomoxir, an FAO inhibitor, and/or platinum-based chemotherapy. CPT1A is overexpressed in HGSOC, correlates with poor overall survival, and is upregulated in HGSOC cells cultured in suspension. CPT1A knockdown promoted anoikis and reduced viability of cells cultured in suspension. HGSOC cells in suspension culture are dependent on CPT1A for mitochondrial activity. In a patient-derived xenograft model of HGSOC, etomoxir significantly inhibited tumor progression. IMPLICATIONS: Targeting FAO in HGSOC to promote anoikis and attenuate dissemination is a potential approach to promote a more durable antitumor response and improve patient outcomes.

Multi-Omic Approaches Identify Metabolic and Autophagy Regulators Important in Ovarian Cancer Dissemination.

High-grade serous ovarian cancers (HGSOCs) arise from exfoliation of transformed cells from the fallopian tube, indicating that survival in suspension, and potentially escape from anoikis, is required for dissemination. We report here the results of a multi-omic study to identify drivers of anoikis escape, including transcriptomic analysis, global non-targeted metabolomics, and a genome-wide CRISPR/Cas9 knockout (GeCKO) screen of HGSOC cells cultured in adherent and suspension settings. Our combined approach identified known pathways, including NOTCH signaling, as well as novel regulators of anoikis escape. Newly identified genes include effectors of fatty acid metabolism, ACADVL and ECHDC2, and an autophagy regulator, ULK1. Knockdown of these genes significantly inhibited suspension growth of HGSOC cells, and the metabolic profile confirmed the role of fatty acid metabolism in survival in suspension. Integration of our datasets identified an anoikis-escape gene signature that predicts overall survival in many carcinomas.

Adiponectin receptor agonist AdipoRon induces apoptotic cell death and suppresses proliferation in human ovarian cancer cells.

We tested the hypothesis that stimulation of adiponectin receptors with the synthetic agonist AdipoRon suppresses proliferation and induces apoptotic death in human high grade serous ovarian tumor cell lines and in ex vivo primary tumors, mediated by activation of 5' AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR). We determined the effect of AdipoRon on high grade serous ovarian tumor cells lines (OVCAR3, OVCAR4, A2780) and ex vivo primary tumor tissue. Western blotting analysis was performed to examine changes in activation of AMPK and mTOR signaling and flow cytometry was utilized to examine changes in cell cycle progression. Immunofluorescence of cleaved caspase-3 positive cells and flow cytometry of annexin V positive cells were used to determine changes in apoptotic response. The CyQUANT proliferation assay was used to assess cell proliferation. AdipoRon treatment increased AMPK phosphorylation (OVCAR3 P = 0.01; A2780 P = 0.02) but did not significantly alter mTOR activity. AdipoRon induced G1 cell cycle arrest in OVCAR3 (+ 12.1%, P = 0.03) and A2780 (+ 12.0%, P = 0.002) cells. OVCAR3 and OVCAR4 cells treated with AdipoRon underwent apoptosis based on cleaved caspase-3 and annexin V staining. AdipoRon treatment resulted in a dose dependent decrease in cell number versus vehicle treatment in OVCAR3 (-61.2%, P < 0.001), OVCAR4 (-79%, P < 0.001), and A2780 (-56.9%, P < 0.001). Ex vivo culture of primary tumors treated with AdipoRon resulted in an increase in apoptosis measured with cleaved caspase-3 immunohistochemistry. AdipoRon induces activation of AMPK and exhibits an anti-tumor effect in ovarian cancer cell lines and primary tumor via a mTOR-independent pathway.

CBX2 identified as driver of anoikis escape and dissemination in high grade serous ovarian cancer.

High grade serous ovarian carcinoma (HGSOC) is often diagnosed at an advanced stage. Chromobox 2 (CBX2), a polycomb repressor complex subunit, plays an oncogenic role in other cancers, but little is known about its role in HGSOC. We hypothesize that CBX2 upregulation promotes HGSOC via induction of a stem-like transcriptional profile and inhibition of anoikis. Examination of Gene Expression Omnibus (GEO) datasets and The Cancer Genome Atlas (TCGA) established that increased CBX2 expression conveyed chemoresistance and worse disease-free and overall survival. In primary HGSOC tumors, we observed CBX2 expression was significantly elevated compared to benign counterparts. In HGSOC cell lines, forced suspension promoted CBX2 expression. Subsequently, CBX2 knockdown inhibited anchorage-independent proliferation and potentiated anoikis-dependent apoptosis. Furthermore, CBX2 knockdown re-sensitized cells to platinum-based chemotherapy. Forced suspension promoted increased ALDH activity and ALDH3A1 expression and CBX2 knockdown led to a decrease in both ALDH activity and ALDH3A1 expression. Investigation of CBX2 expression on a HGSOC tissue microarray revealed CBX2 expression was apparent in both primary and metastatic tissues. CBX2 is an important regulator of stem-ness, anoikis escape, HGSOC dissemination, and chemoresistance and potentially serves as a novel therapeutic target.

Statistical performance of image cytometry for DNA, lipids, cytokeratin, & CD45 in a model system for circulation tumor cell detection.

Detection of circulating tumor cells (CTCs) in a blood sample is limited by the sensitivity and specificity of the biomarker panel used to identify CTCs over other blood cells. In this work, we present Bayesian theory that shows how test sensitivity and specificity set the rarity of cell that a test can detect. We perform our calculation of sensitivity and specificity on our image cytometry biomarker panel by testing on pure disease positive (D+ ) populations (MCF7 cells) and pure disease negative populations (D- ) (leukocytes). In this system, we performed multi-channel confocal fluorescence microscopy to image biomarkers of DNA, lipids, CD45, and Cytokeratin. Using custom software, we segmented our confocal images into regions of interest consisting of individual cells and computed the image metrics of total signal, second spatial moment, spatial frequency second moment, and the product of the spatial-spatial frequency moments. We present our analysis of these 16 features. The best performing of the 16 features produced an average separation of three standard deviations between D+ and D- and an average detectable rarity of ∼1 in 200. We performed multivariable regression and feature selection to combine multiple features for increased performance and showed an average separation of seven standard deviations between the D+ and D- populations making our average detectable rarity of ∼1 in 480. Histograms and receiver operating characteristics (ROC) curves for these features and regressions are presented. We conclude that simple regression analysis holds promise to further improve the separation of rare cells in cytometry applications. © 2017 International Society for Advancement of Cytometry.

Progestin treatment decreases CD133+ cancer stem cell populations in endometrial cancer.

OBJECTIVES: Endometrial cancer is a hormonally responsive malignancy. Response to progestins is associated with estrogen receptor (ER) and progesterone receptor (PR) status. CD133 is a marker of endometrial cancer stem cells. We postulated that CD133+ cells express ER and PR and that progestin therapy differentially regulates CD133+ cells. METHODS: The Ishikawa (ER/PR positive) and KLE (ER/PR negative) cell lines were examined for the presence of CD133 populations. Cell lines were treated with 30.4µM medroxyprogesterone 17-acetate (MPA) for 6days. After treatment, cell counts, apoptosis assays and CD133+ populations were examined. In a clinical project, we identified 12 endometrial cancer patients who were treated with progestin drugs at our institution. Using immunohistochemistry, CD133, ER, PR, and androgen receptor (AR) expression was scored and evaluated for change over time on serial biopsies. RESULTS: CD133+ populations were identified in Ishikawa and KLE cell lines. MPA treatment resulted in a significant reduction in the percentage of live cells (Ishikawa, P=0.036; KLE, P=0.0002), significant increase in apoptosis (Ishikawa, P=0.01; KLE, P=0.0006) and significant decrease in CD133+ populations (Ishikawa, P<0.0001; KLE, P=0.0001). ER, PR, AR and CD133 were present in 96.4%, 96.4%, 89.3% and 100% of patient samples respectively. Paralleling the in vitro results, CD133 expression decreased in patients who had histologic response to progestin treatment. CONCLUSION: CD133+ populations decreased after treatment with MPA in an in vitro model and in patients responding to treatment with progestins. Progestin treatment differentially decreases CD133+ cells.

Cytokeratin 5-Positive Cells Represent a Therapy Resistant subpopulation in Epithelial Ovarian Cancer.

OBJECTIVE: Cytokeratin 5 (CK5) is an epithelial cell marker implicated in stem and progenitor cell activity in glandular reproductive tissues and endocrine and chemotherapy resistance in estrogen receptor (ER)(+) breast cancer. The goal of this study was to determine the prevalence of CK5 expression in ovarian cancer and the response of CK5(+) cell populations to cisplatin therapy. MATERIALS AND METHODS: Cytokeratin 5 expression was evaluated in 2 ovarian tissue microarrays, representing 137 neoplasms, and 6 ovarian cancer cell lines. Cell lines were treated with IC(50) (half-maximal inhibitory concentration) cisplatin, and the prevalence of CK5(+) cells pretreatment and posttreatment was determined. Proliferation of CK5(+) versus CK5(-) cell populations was determined using 5-bromo-2'-deoxyuridine incorporation. Chemotherapy-induced apoptosis in CK5(+) versus CK5(-) cells was measured using immunohistochemical staining for cleaved caspase-3. RESULTS: Cytokeratin 5 was expressed in 39.3% (42 of 107) of epithelial ovarian cancers with a range of 1% to 80% positive cells. Serous and endometrioid histologic subtypes had the highest percentage of CK5(+) specimens. Cytokeratin 5 expression correlated with ER positivity (38 of 42 CK5(+) tumors were also ER(+)). Cytokeratin 5 was expressed in 5 of 6 overall and 4 of 4 ER(+) epithelial ovarian cancer cell lines ranging from 2.4% to 52.7% positive cells. Cytokeratin 5(+) compared with CK5(-) cells were slower proliferating. The prevalence of CK5(+) cells increased after 48-hour cisplatin treatment in 4 of 5 cell lines tested. Cytokeratin 5(+) ovarian cancer cells compared with CK5(-) ovarian cancer cells were more resistant to cisplatin-induced apoptosis. CONCLUSIONS: Cytokeratin 5 is expressed in a significant proportion of epithelial ovarian cancers and represents a slower proliferating chemoresistant subpopulation that may warrant cotargeting in combination therapy.

Specificity and prognostic validation of a polyclonal antibody to detect Six1 homeoprotein in ovarian cancer.

OBJECTIVE: The presence of Six1 mRNA gene portends a poor prognosis in ovarian cancer. We describe validation of a Six1 specific antibody and evaluate its association with tumorigenicity and prognosis in ovarian cancer. METHODS: A Six1 antibody (Six1cTerm) was raised to residues downstream of the Six1 homeodomain, representing its unique C-terminus as compared to other Six family members. Cells were transfected with Six1-Six6 and Western blot was performed to demonstrate Six1 specificity. Ovarian cancer cell lines were analyzed for Six1 mRNA and Six1cTerm and tumorigenicity was evaluated. Ovarian cancer tissue microarrays (OTMA) were analyzed for Six1cTerm by immunohistochemistry and scored by two blinded observers. The metastatic tumors of 15 stage IIIC high grade serous ovarian cancers were analyzed with Six1 mRNA and Six1cTerm and expression was compared to clinical factors and survival. RESULTS: The Six1cTerm antibody is specific for Six1. Cell line tumorigenicity in SCID mice correlates with Six1 levels both by mRNA(p=0.001, Mann-Whitney U test) and by protein (presence vs. absence, p=0.05 Fischer's Exact test). Six1 protein was present in up to 54% of OTMA specimens. Six1 protein expression in omental/peritoneal metastases correlated with worsened survival in a sample (n=15) of high grade serous stage IIIC ovarian cancers (p=0.001). CONCLUSIONS: The Six1cTerm antibody is specific and able to detect Six1 in cell lines and tumor tissue. Six1 protein detection is common in ovarian cancer and is associated with tumorigenicity and poor prognosis in this group of patient samples. Six1cTerm antibody should be further validated as prognostic tool.

TRAIL receptor signaling regulation of chemosensitivity in vivo but not in vitro.

BACKGROUND: Signaling by Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) and Fas ligand (FasL) has been proposed to contribute to the chemosensitivity of tumor cells treated with various other anti-cancer agents. However, the importance of these effects and whether there are differences in vitro and in vivo is unclear. METHODOLOGY/PRINCIPAL FINDINGS: To assess the relative contribution of death receptor pathways to this sensitivity and to determine whether these effects are intrinsic to the tumor cells, we compared the chemosensitivity of isogenic BJAB human lymphoma cells where Fas and TRAIL receptors or just TRAIL receptors were inhibited using mutants of the adaptor protein FADD or by altering the expression of the homeobox transcription factor Six1. Inhibition of TRAIL receptors did not affect in vitro tumor cell killing by various anti-cancer agents indicating that chemosensitivity is not significantly affected by the tumor cell-intrinsic activation of death receptor signaling. However, selective inhibition of TRAIL receptor signaling caused reduced tumor regression and clearance in vivo when tested in a NOD/SCID mouse model. CONCLUSIONS: These data show that TRAIL receptor signaling in tumor cells can determine chemosensitivity in vivo but not in vitro and thus imply that TRAIL resistance makes tumors less susceptible to conventional cytotoxic anti-cancer drugs as well as drugs that directly target the TRAIL receptors.

Protein kinase C inhibitor Gö6976 augments caffeine-induced reversal of chemoresistance to cis-diamminedichloroplatinum-II (CDDP) in a human ovarian cancer model.

OBJECTIVES: Novel strategies for the treatment of chemoresistant ovarian cancer are needed. Caffeine and related compounds have been shown to over-ride G2/M arrest in ovarian cancer cells, increasing toxicity to chemotherapy. Newer compounds have been developed which may have the same effect as and exhibit synergism with caffeine, allowing the use of lower doses. METHODS: We investigated the effects of caffeine and Gö6976 in the presence of CDDP in the SKOV3 and A2780 cell lines using proliferation, cell-cycle analysis, apoptosis, and AKT expression. RESULTS: Proliferation of cancer cells was decreased in a dose-dependent manner with caffeine and CDDP, but doses of caffeine required for significant inhibition were higher than that achievable in patients. Gö6976, a global PKC inhibitor with G2/M over-ride capability similar to caffeine, when combined with caffeine and CDDP at doses below that required for cell-cycle over-ride produced the growth inhibitory effects of a ten-fold higher caffeine concentration in both cell lines. CDDP induced G2/M arrest was significantly abrogated by caffeine but not by Gö6976 alone and no additional effect was seen on G2/M over-ride by the addition of Gö6976 to caffeine. Addition of Gö6976 to caffeine and CDDP did increase apoptosis but without altering phospho-AKT. CONCLUSIONS: Gö6976, when added to caffeine at doses below that required for cell-cycle over-ride, augments caffeine in overcoming CDDP resistance in this experimental system. G2/M over-ride is not the mechanism underlying the inhibition of proliferation. An AKT-independent apoptotic mechanism may be responsible.

Photoprotective effects of bucillamine against UV-induced damage in an SKH-1 hairless mouse model.

UVB exposure of skin results in various biologic responses either through direct or indirect damage to DNA and non-DNA cellular targets via the formation of free radicals, reactive oxygen species (ROS) and inflammation. Bucillamine [N-(2-mercapto-2-methylpropionyl)-l-cysteine] is a cysteine-derived compound that can replenish endogenous glutathione due to its two donatable thiol groups, and functions as an antioxidant. In this study, we investigated the effects of bucillamine on UVB-induced photodamage using the SKH-1 hairless mouse model. We have demonstrated that UVB exposure (two consecutive doses, 230 mJ cm(-2)) on the dorsal skin of SKH-1 mice induced inflammatory responses (edema, erythema, dermal infiltration of leukocytes, dilated blood vessels) and p53 activation as early as 6 h after the last UVB exposure. Bucillamine pretreatment (20 mg kg(-1) of body weight, administered subcutaneously) markedly attenuated UVB-mediated inflammatory responses and p53 activation. We have also demonstrated that the stabilization and upregulation of p53 by UVB correlated with phosphorylation of Ser-15 and Ser-20 residues of p53 protein and that bucillamine pretreatment attenuated this effect. We propose that bucillamine has potential to be effective as a photoprotective agent for the management of pathologic conditions elicited by UV exposure.

Six1 overexpression in ovarian carcinoma causes resistance to TRAIL-mediated apoptosis and is associated with poor survival.

Tumorigenesis can arise from inappropriate activation of developmental genes in mature tissues. Here, we show that the developmental regulator Six1 is overexpressed in ovarian carcinoma cell lines (OCC) compared with normal ovarian surface epithelium. As observed in other cancers, Six1 overexpression in OCC leads to increased A-type cyclin expression and increased proliferation. In addition, Six1 overexpression renders OCC resistant to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis, and Six1 knockdown in the TRAIL-resistant SKOV3 ovarian carcinoma line dramatically sensitizes the cells to TRAIL. Because inactivation of the TRAIL response has been linked to metastasis, and because antibodies and recombinant ligand that activate the TRAIL pathway are currently in clinical trials against ovarian carcinoma, we screened normal ovarian and carcinoma specimens for Six1 mRNA. Six1 was overexpressed in 50% of the early-stage (stage I) and 63% of the late-stage (stages II, III, and IV) ovarian carcinomas examined, with late-stage carcinomas expressing approximately 3-fold higher Six1 mRNA levels on average compared with early-stage tumors. Importantly, in patients with late-stage disease, high Six1 expression was associated with significantly shortened survival (P = 0.0015). These data suggest that Six1 may contribute to ovarian epithelial carcinogenesis by simultaneously increasing proliferation and decreasing TRAIL-mediated apoptosis and imply that Six1 may be an important determinant of TRAIL therapy response that should be considered in patient selection for TRAIL-related clinical trials.

Molecular cloning and baculovirus expression of the rabbit corneal aldehyde dehydrogenase (ALDH1A1) cDNA.

Most mammalian species express high concentrations of ALDH3A1 in corneal epithelium with the exception of the rabbit, which expresses high amounts of ALDH1A1 rather than ALDH3A1. Several hypotheses that involve catalytic and/or structural functions have been postulated regarding the role of these corneal ALDHs. The aim of the present study was to characterize the biochemical properties of the rabbit ALDH1A1. We have cloned and sequenced the rabbit ALDH1A1 cDNA, which is 2,073 bp in length (excluding the poly(A+) tail), and has 5' and 3' nontranslated regions of 46 and 536 bp, respectively. This ALDH1A1 cDNA encodes a protein of 496 amino acids (Mr = 54,340) that is: 86-91% identical to mammalian ALDH1A1 proteins, 83-85% identical to phenobarbital-inducible mouse and rat ALDH1A7 proteins, 84% identical to elephant shrew ALDH1A8 proteins (eta-crystallins), 69-73% identical to vertebrate ALDH1A2 and ALDH1A3 proteins, 65% identical to scallop ALDH1A9 protein (omega-crystallin), and 55-57% to cephalopod ALDH1C1 and ALDH1C2 (omega-crystallins). Recombinant rabbit ALDH1A1 protein was expressed using the baculovirus system and purified to homogeneity with affinity chromatography. We found that rabbit ALDH1A1 is catalytically active and efficiently oxidizes hexanal (Km = 3.5 microM), 4-hydroxynonenal (Km = 2.1 microM) and malondialdehyde (Km = 14.0 microM), which are among the major products of lipid peroxidation. Similar kinetic constants were observed with the human recombinant ALDH1A1 protein, which was expressed and purified using similar experimental conditions. These data suggest that ALDH1A1 may contribute to corneal cellular defense against oxidative damage by metabolizing toxic aldehydes produced during UV-induced lipid peroxidation.

Involvement of the electrophile responsive element and p53 in the activation of hepatic stellate cells as a response to electrophile menadione.

The cytotoxic effects of menadione and hydrogen peroxide were examined in two hepatic stellate cell lines derived from normal or cirrhotic rat liver. The cirrhotic fat-storing cells (CFSC) were found more resistant than the normal fat-storing cells (NFSC) to menadione cytotoxicity. No significant differences were observed in hydrogen peroxide toxicity in these two cell lines. Although protein levels and enzymatic activities of catalase, Cu,Zn-SOD, Mn-SOD, and NADPH cytochrome c reductase were similar in these cell lines, 20-fold increases of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzymatic activity and protein levels were detected in CFSC compared to those of NFSC. Gel mobility shift assays and functional analysis using transient transfection experiments indicated the involvement of the electrophile responsive element (EPRE) in the up-regulation of the NQO1 expression. Antibody supershift analysis revealed that, although Nrf2 is a member of the EPRE-binding complex in both NFSC and CFSC, Nrf1 was identified as a part of the protein/DNA complex only in CFSC. Expression of p53 tumor suppressor gene was found in higher levels in CFSC than in NFSC. We conclude that activation of the EPRE-signaling pathway, which up-regulates several phase II genes and affects p53 stabilization, may offer resistance to hepatic stellate cells against oxidative damage during hepatic injury. This resistance may be a part of the activation process of the hepatic stellate cells and could contribute to their increased proliferation and production of extracellular matrix.