Comparative analysis of response to treatments and molecular features of tumor-derived organoids versus cell lines and PDX derived from the same ovarian clear cell carcinoma.

BACKGROUND: In the era of personalized medicine, the establishment of preclinical models of cancer that faithfully recapitulate original tumors is essential to potentially guide clinical decisions. METHODS: We established 7 models [4 cell lines, 2 Patient-Derived Tumor Organoids (PDTO) and 1 Patient-Derived Xenograft (PDX)], all derived from the same Ovarian Clear Cell Carcinoma (OCCC). To determine the relevance of each of these models, comprehensive characterization was performed based on morphological, histological, and transcriptomic analyses as well as on the evaluation of their response to the treatments received by the patient. These results were compared to the clinical data. RESULTS: Only the PDX and PDTO models derived from the patient tumor were able to recapitulate the patient tumor heterogeneity. The patient was refractory to carboplatin, doxorubicin and gemcitabine, while tumor cell lines were sensitive to these treatments. In contrast, PDX and PDTO models displayed resistance to the 3 drugs. The transcriptomic analysis was consistent with these results since the models recapitulating faithfully the clinical response grouped together away from the other classical 2D cell culture models. We next investigated the potential of drugs that have not been used in the patient clinical management and we identified the HDAC inhibitor belinostat as a potential effective treatment based on PDTO response. CONCLUSIONS: PDX and PDTO appear to be the most relevant models, but only PDTO seem to present all the necessary prerequisites for predictive purposes and could constitute relevant tools for therapeutic decision support in the context of these particularly aggressive cancers refractory to conventional treatments.

Network-Based Integration of Multi-Omics Data Identifies the Determinants of miR-491-5p Effects.

The identification of miRNAs' targets and associated regulatory networks might allow the definition of new strategies using drugs whose association mimics a given miRNA's effects. Based on this assumption we devised a multi-omics approach to precisely characterize miRNAs' effects. We combined miR-491-5p target affinity purification, RNA microarray, and mass spectrometry to perform an integrated analysis in ovarian cancer cell lines. We thus constructed an interaction network that highlighted highly connected hubs being either direct or indirect targets of miR-491-5p effects: the already known EGFR and BCL2L1 but also EP300, CTNNB1 and several small-GTPases. By using different combinations of specific inhibitors of these hubs, we could greatly enhance their respective cytotoxicity and mimic the miR-491-5p-induced phenotype. Our methodology thus constitutes an interesting strategy to comprehensively study the effects of a given miRNA. Moreover, we identified targets for which pharmacological inhibitors are already available for a clinical use or in clinical trials. This study might thus enable innovative therapeutic options for ovarian cancer, which remains the leading cause of death from gynecological malignancies in developed countries.

The lncRNA 'UCA1' modulates the response to chemotherapy of ovarian cancer through direct binding to miR-27a-5p and control of UBE2N levels.

Ovarian cancer (OC) is the leading cause of death in patients with gynecologic cancers. Due to late diagnosis and resistance to chemotherapy, the 5-year survival rate in patients with OC is below 40%. We observed that UCA1, a lncRNA previously reported to play an oncogenic role in several malignancies, is overexpressed in the chemoresistant OC cell line OAW42-R compared to their chemotherapy-sensitive counterpart OAW42. Additionally, UCA1 overexpression was related to poor prognosis in two independent patient cohorts. Currently, the molecular mechanisms through which UCA1 acts in OC are poorly understood. We demonstrated that downregulation of the short isoform of UCA1 sensitized OC cells to cisplatin and that UCA1 acted as competing endogenous RNA to miR-27a-5p. Upon UCA1 downregulation, miR-27a-5p downregulated its direct target UBE2N leading to the upregulation of BIM, a proapoptotic protein of the Bcl2 family. The upregulation of BIM is the event responsible for the sensitization of OC cells to cisplatin. In order to model response to therapy in patients with OC, we used several patient-derived organoid cultures, a model faithfully mimicking patient's response to therapy. Inhibition of UBE2N sensitized patient-derived organoids to platinum salts. In conclusion, response to treatment in patients with OC is regulated by the UCA1/miR-27a-5p/UBE2N axis, where UBE2N inhibition could potentially represent a novel therapeutic strategy to counter chemoresistance in OC.

Bim, Puma and Noxa upregulation by Naftopidil sensitizes ovarian cancer to the BH3-mimetic ABT-737 and the MEK inhibitor Trametinib.

Ovarian cancer represents the first cause of mortality from gynecologic malignancies due to frequent chemoresistance occurrence. Increasing the [BH3-only Bim, Puma, Noxa proapoptotic]/[Bcl-xL, Mcl-1 antiapoptotic] proteins ratio was proven to efficiently kill ovarian carcinoma cells and development of new molecules to imbalance Bcl-2 member equilibrium are strongly required. Drug repurposing constitutes an innovative approach to rapidly develop therapeutic strategies through exploitation of established drugs already approved for the treatment of noncancerous diseases. This strategy allowed a renewed interest for Naftopidil, an α1-adrenergic receptor antagonist commercialized in Japan for benign prostatic hyperplasia. Naftopidil was reported to decrease the incidence of prostate cancer and its derivative was described to increase BH3-only protein expression in some cancer models. Based on these arguments, we evaluated the effects of Naftopidil on ovarian carcinoma and showed that Naftopidil reduced cell growth and increased the expression of the BH3-only proteins Bim, Puma and Noxa. This effect was independent of α1-adrenergic receptors blocking and involved ATF4 or JNK pathway depending on cellular context. Finally, Naftopidil-induced BH3-only members sensitized our models to ABT-737 and Trametinib treatments, in vitro as well as ex vivo, in patient-derived organoid models.

Functional miRNA Screening Identifies Wide-ranging Antitumor Properties of miR-3622b-5p and Reveals a New Therapeutic Combination Strategy in Ovarian Tumor Organoids.

Novel therapeutic strategies are urgently required for the clinical management of chemoresistant ovarian carcinoma, which is the most lethal of the gynecologic malignancies. miRNAs hold promise because they play a critical role in determining the cell phenotype by regulating several hundreds of targets, which could constitute vulnerabilities of cancer cells. A combination of gain-of-function miRNA screening and real-time continuous cell monitoring allows the identification of miRNAs with robust cytotoxic effects in chemoresistant ovarian cancer cells. Focusing on miR-3622b-5p, we show that it induces apoptosis in several ovarian cancer cell lines by both directly targeting Bcl-xL and EGFR-mediating BIM upregulation. miR-3622b-5p also sensitizes cells to cisplatin by inhibiting Bcl-xL in ovarian cancer cell lines escaping BIM induction. miR-3622b-5p also exerts antimigratory capacities by targeting both LIMK1 and NOTCH1. These wide-ranging antitumor properties of miR-3622b-5p in ovarian cancer cells are mimicked by the associations of pharmacologic inhibitors targeting these proteins. The combination of an EGFR inhibitor together with a BH3-mimetic molecule induced a large decrease in cell viability in a panel of ovarian cancer cell lines and several ovarian patient-derived tumor organoids, suggesting the value of pursuing such a combination therapy in ovarian carcinoma. Altogether, our work highlights the potential of phenotype-based miRNA screening approaches to identify lethal interactions which might lead to new drug combinations and clinically applicable strategies.

Inhibition of store-operated channels by carboxyamidotriazole sensitizes ovarian carcinoma cells to anti-BclxL strategies through Mcl-1 down-regulation.

The anti-apoptotic proteins Bcl-xL and Mcl-1 have been identified to play a pivotal role in apoptosis resistance in ovarian cancer and constitute key targets for innovative therapeutic strategies. Although BH3-mimetics (i.e. ABT-737) potently inhibit Bcl-xL activity, targeting Mcl-1 remains a hurdle to the success of these strategies. Calcium signaling is profoundly remodeled during carcinogenesis and was reported to activate the signaling pathway controlling Mcl-1 expression. In this context, we investigated the effect of carboxyamidotriazole (CAI), a calcium channel inhibitor used in clinical trials, on Mcl-1 expression. CAI had an anti-proliferative effect on ovarian carcinoma cell lines and strongly down-regulated Mcl-1 expression. It inhibited store-operated calcium entry (SOCE) and Mcl-1 translation through mTORC1 deactivation. Moreover, it sensitized ovarian carcinoma cells to anti-Bcl-xL strategies as their combination elicited massive apoptosis. Its effect on mTORC1 and Mcl-1 was mimicked by the potent SOCE inhibitor, YM58483, which also triggered apoptosis when combined with ABT-737. As a whole, this study suggests that CAI sensitizes to anti-Bcl-xL strategies via its action on Mcl-1 translation and that modulation of SOCE could extend the therapeutic arsenal for treatment of ovarian carcinoma.

The mTORC1/2 Inhibitor AZD8055 Strengthens the Efficiency of the MEK Inhibitor Trametinib to Reduce the Mcl-1/[Bim and Puma] ratio and to Sensitize Ovarian Carcinoma Cells to ABT-737.

The identification of novel therapeutic strategies is an important urgent requirement for the clinical management of ovarian cancer, which remains the leading cause of death from gynecologic cancer. Several studies have shown that the antiapoptotic proteins Bcl-xL and Mcl-1, as well as the proapoptotic protein Bim, are key elements to be modulated to kill ovarian cancer cells. Pharmacologic inhibition of Bcl-xL is possible by using BH3-mimetic molecules like ABT-737. However, inhibition of Mcl-1 and/or promotion of its BH3-only partners (including Bim, Puma, and Noxa) remains a challenge that may be achieved by modulating the signaling pathways upstream. This study sought whether AZD8055-induced mTOR inhibition and/or trametinib-induced MEK inhibition could modulate Mcl-1 and its partners to decrease the Mcl-1/BH3-only ratio and thus sensitize various ovarian cancer cell lines to ABT-737. AZD8055 treatment inhibited Mcl-1 and increased Puma expression but did not induce massive apoptosis in combination with ABT-737. In contrast, trametinib, which decreased the Mcl-1/BH3-only protein ratio by upregulating Puma and dephosphorylated active Bim, sensitized IGROV1-R10 and OVCAR3 cells to ABT-737. Adding AZD8055 to trametinib further reduced the Mcl-1/BH3-only protein ratio and triggered apoptosis without ABT-737 in IGROV1-R10 cells. Moreover, the AZD8055/trametinib association highly sensitized all cell lines including SKOV3 to ABT-737, the induced dephosphorylated Bim being crucial in this sensitization. Finally, the three-drug combination was also very efficient when replacing AZD8055 by the pan-Akt inhibitor MK-2206. This study thus proposes original multitargeted strategies and may have important implications for the design of novel approaches for ovarian cancer treatment. Mol Cancer Ther; 16(1); 102-15. ©2016 AACR.

Calcium signals inhibition sensitizes ovarian carcinoma cells to anti-Bcl-xL strategies through Mcl-1 down-regulation.

Ovarian carcinoma is the leading cause of death from gynecologic cancer in the developed world and is characterized by acquired chemoresistance leading to an overall 5-year survival rate of about 30 %. We previously showed that Bcl-xL and Mcl-1 cooperatively protect platinum-resistant ovarian cancer cells from apoptosis. Despite BH3-mimetics represent promising drugs to target Bcl-xL, anti-Mcl-1 strategies are still in pre-clinical studies and required new investigations. Calcium is a universal second messenger and dysregulation of calcium signal is often observed during carcinogenesis. As change in cytosolic free calcium concentration [Ca(2+)]i is known to control the fate of the cell by regulating Bcl-2 family members, we wonder if calcium signal could impact on Mcl-1 expression and if its pharmacological inhibition could be useful to sensitize ovarian carcinoma cells to anti-Bcl-xL strategies. We therefore studied the effect of different calcium signals inhibitors in ovarian carcinoma cell lines SKOV3 and IGROV1-R10 and analysed their effects on proliferation and Mcl-1 expression. We also exposed these cells to these inhibitors in combination with anti-Bcl-xL strategies (siRNA or BH3-mimetic: ABT-737). We found that calcium signaling regulates Mcl-1 through translational events and a calmodulin-mediated pathway. BAPTA-AM and calmodulin inhibitor combination with ABT-737 leads to apoptosis, a process that is reversed by Mcl-1 enforced expression. As Mcl-1 represents a crucial hurdle to the success of chemotherapy, these results could open to new area of investigation using calcium modulators to directly or indirectly target Mcl-1 and thus efficiently sensitize ovarian carcinoma cells to anti-Bcl-xL strategies.

Identification of predictive factors of response to the BH3-mimetic molecule ABT-737: an ex vivo experiment in human serous ovarian carcinoma.

Ovarian cancers are addicted to Bcl-xL and Mcl-1, antiapoptotic members of the Bcl-2 family. Bcl-xL can be inhibited by the BH3-mimetic ABT-737. In vitro, ABT-737 can induce apoptosis of cancer cells, and its activity is potentiated by Mcl-1 inactivation. Herein, we assessed the sensitivity of human ovarian tumor nodes to ABT-737 when combined with carboplatin, which can indirectly inhibit Mcl-1. Fresh samples from 25 patients with high-grade serous ovarian cancer (HGSOC) who were chemo-naïve and had undergone surgery were prospectively exposed ex vivo to ABT-737 ± carboplatin. The treatment effect was studied on sliced tumor nodes by assessment of cleaved-caspase 3 immunostaining. We also studied the association between baseline Bcl-2 family protein expression (via immunohistochemistry) and the response of nodes to treatment. ABT-737 induced apoptosis as a single agent but its efficacy was not improved by the addition of carboplatin. Bim was frequently expressed (20/25) and its absence or low expression was associated with the absence of response to ABT-737, p value = 0.019 by Fisher's test and sensitivity = 93%, (95% confidence interval, 66-100). Moreover, we observed that in tumors in which Bim was expressed, a low expression of phospho-Erk1/2 or Mcl-1 improved the proportion of responses. This pilot study showed that ABT-737 has promise as monotherapy for HGSOC in a specific subgroup of tumors. Bim, Mcl-1, and phospho-Erk1/2 appeared to be relevant biomarkers that could be used for the selection of patients in the design of clinical trials using Navitoclax (an orally available compound related to ABT-737).

PI3K/mTOR dual inhibitor NVP-BEZ235 decreases Mcl-1 expression and sensitizes ovarian carcinoma cells to Bcl-xL-targeting strategies, provided that Bim expression is induced.

We previously showed that Bcl-xL and Mcl-1 cooperatively protect platinum-resistant ovarian cancer cells from apoptosis. Here we assessed the anticancer potential of combining ABT-737-induced inhibition of Bcl-xL with Mcl-1 inhibition via PI3K/Akt/mTOR pathway disruption using NVP-BEZ235. NVP-BEZ235 inhibited cell proliferation without inducing apoptosis. It strongly repressed Mcl-1 expression and induced Puma expression in both cell lines tested while differentially modulating Bim between the two. Interestingly, NVP-BEZ235 efficiently sensitized ovarian carcinoma cells to ABT-737, provided that Bim expression was induced. Moreover, inhibiting the ERK1/2 pathway restored Bim expression and sensitized low Bim-expressing cancer cells to the BEZ235/ABT-737 treatment.

Sensitization of ovarian carcinoma cells to Bcl-xL-targeting strategies through indirect modulation of Mcl-1 activity by MR22388, a molecule of the tripentone family.

BACKGROUND: Our work has been carried out in the context of the therapeutic failure in ovarian carcinoma, which remains the leading cause of death by gynecologic malignancy. In these tumours, recurrence and subsequent acquired chemoresistance constitute major hurdles to successful therapy. Here we studied the interest of a member of the tripentone chemical family, MR22388, for the treatment of chemoresistant ovarian cancer cells. FINDINGS: MR22388 activity has been assessed in vitro on cisplatin-resistant (SKOV3 and IGROV1-R10) ovarian cancer cell lines by conventional analysis, alone or combined to a BH3-mimetic molecule, ABT-737. MR22388 exerts its activity on cisplatin resistant cells, and we showed that it induces a decrease of the Mcl-1 anti-apoptotic protein expression. Considering our previous work demonstrating that the efficiency of Bcl-xL targeting strategies is conditioned to the concomitant inhibition of Mcl-1 we studied the interest of the association of this MR22388 with ABT-737, and showed that this combination was highly cytotoxic in chemoresistant cells. CONCLUSIONS: This work thus opens new perspectives for the use of this promising molecule for the treatment of highly chemoresistant ovarian cancer cells and for sensitization of emerging Bcl-xL targeting strategies such as the use of BH3-mimetic molecules.

Quality of life in long-term cervical cancer survivors: a population-based study.

OBJECTIVES: To assess long-term quality of life (QOL) in cervical cancer survivors (CCSs), 5, 10, and 15 years after diagnosis. METHODS: In a cross-sectional population-based study, CCSs diagnosed in 1990, 1995, or 2000 were randomly selected from 3 tumor registries in France. Healthy controls were randomly selected from electoral rolls, stratifying on age group and residence area. Five QOL questionnaires (SF-36, EORTC QLQ-C30, the cervical cancer-specific module (EORTC QLQ-CX24), the MFI fatigue questionnaire, the STAI for anxiety) and a life condition questionnaire were used. Analysis of variance was used to compare QOL scores of survivors by period of diagnosis (5, 10, and 15 years) with those of controls and according to treatment modality, adjusted for socio-demographic data. RESULTS: A total of 173 localized CCSs (42% treated with surgery alone and 58% with a combination of treatments) and 594 controls participated in the study. Compared with controls, CCSs expressed globally similar good QOL, except for impaired psychoemotional domains in 15-year survivors (p<0.01). Worsening of some symptoms was observed over time, 15-year survivors in particular reported significantly more lymphedema than 5-year (p=0.0009) and 10-year CCSs (p=0.002). Compared with CCSs treated by surgery alone, QOL of CCSs who received radiotherapy was significantly more affected in terms of cervical cancer specific problems, such as sexual dysfunction (p=0.002), voiding and abdominal symptoms (p=0.01), and lymphedema (p=0.01). CONCLUSIONS: Even after 15 years, QOL of CCSs is impacted in psychological domains, compared with healthy controls. Among CCSs, women treated by adjuvant radiotherapy expressed more physical sequelae.

Health-related quality of life among long-term survivors of colorectal cancer: a population-based study.

BACKGROUND: The number of long-term colorectal cancer survivors is increasing. Cancer and its treatment can cause physical and psychological complications, but little is known about how it impacts quality of life (QOL) over the long term-5, 10, and 15 years after diagnosis. METHODS: Cancer survivors were randomly selected from three tumor registries in France, diagnosed in 1990 (±1 year), 1995 (±1 year), and 2000 (±1 year). Controls were randomly selected from electoral rolls, stratifying on gender, age group, and residence area. Participants completed two QOL questionnaires, a fatigue questionnaire, an anxiety questionnaire, and a life conditions questionnaire. An analysis of variance was used to compare QOL scores of cancer survivors by period of diagnosis (5, 10, and 15 years) with those of controls, adjusted for sociodemographic data and comorbidities. RESULTS: We included 344 colon cancer and 198 rectal cancer survivors and 1,181 controls. In a global analysis, survivors reported a statistically and clinically significant lower score in social functioning 5 years after diagnosis and higher scores in diarrhea symptoms 5 and 10 years after diagnosis. In subgroup analyses, rectal cancer affected QOL in the physical dimensions at 5 years and in the fatigue dimensions at 5 and 10 years. CONCLUSION: Survivors of colorectal cancer may experience the effects of cancer and its treatment up to 10 years after diagnosis, particularly for rectal cancer. Clinicians, psychologists, and social workers must pay special attention to rectal cancer survivors to improve overall management.

Long-term quality of life after breast cancer: a French registry-based controlled study.

Population-based studies on quality of life (QOL) of long-term breast cancer survivors are quite recent and insufficient attention has been paid to the effect of time since diagnosis. We compared long-term QOL of population-based breast cancer survivors 5, 10, and 15 years after diagnosis with that of healthy controls. Breast cancer survivors were randomly selected from three population-based cancer registries (Bas-Rhin, Calvados and Doubs, France) along with healthy controls, stratified for age and place of residence, randomly selected from electoral rolls. Participants completed five self-administered questionnaires: the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30), Short Form-36 (SF-36), Spielberger State-Trait Anxiety Inventory (STAI), Multidimensional Fatigue Inventory (MFI) and a life conditions questionnaire. An analysis of variance was used to compare QOL scores of breast cancer survivors by period (5, 10, or 15 years) of diagnosis with those of controls, adjusted for sociodemographic data and comorbidities. Six hundred and fifty-two cases and 1,188 controls participated in the study. For many QOL scales, scores were significantly different between cancer survivors and controls. A clinically significant difference was evidenced for the fatigue scales, the SF36 physical functioning, role-physical, and role-emotional scales, with more favorable results for controls. Differences decreased with time and 15-year cancer survivors were generally not different from controls. Scores were particularly influenced by age and mean household income. More efforts should be made, specifically during the first 5 to 10 years after diagnosis, to help women with breast cancer to overcome their impairment in QOL.

Downregulation of Bcl-xL and Mcl-1 is sufficient to induce cell death in mesothelioma cells highly refractory to conventional chemotherapy.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited response to platinum-based chemotherapy. Several lines of evidence support a role for the anti-apoptotic protein Bcl-x(L) in MPM chemoresistance. Since it has been recently suggested that Mcl-1 cooperates with Bcl-x(L) for protection against cell death, we investigated the response of mesothelioma cell lines to the downregulation of Bcl-x(L) (alone or in combination with cisplatin) and the potential interest of its concomitant inhibition with that of Mcl-1. Using RNA interference, we showed that Bcl-x(L) depletion sensitized two highly chemoresistant mesothelioma cell lines to cisplatin and that under this treatment, one cell line, MSTO-211H, displayed an apoptotic type of cell death, whereas the other, NCI-H28, evidenced mainly necrotic-type cell death. Otherwise, the inhibition of Mcl-1 by cisplatin may contribute to this induction of cell death observed after Bcl-x(L) downregulation. Strikingly, we observed that the simultaneous inhibition of Bcl-x(L) and Mcl-1 using small interfering RNA (siRNA) induced a massive cell death in the absence of chemotherapy and was sufficient to avoid escape to treatment in MSTO-211H cells. In NCI-H28, the addition of a low cisplatin concentration allowed to impede the long-term recovery observed after treatment by the siRNA combination. Together, these findings provide a strong molecular basis for the clinical evaluation of therapies targeting both Bcl-x(L) and Mcl-1, alone or in combination with conventional chemotherapy, for the treatment of MPM.