Light‐Triggered Antibody‐Photosensitizer Conjugates Disrupt P‐Glycoprotein Function to Reverse Multidrug Resistance

Aim: ATP-binding cassette (ABC) transporters are proteins responsible for the efflux of drug molecules from cancer cells, reducing the efficacy of anti-cancer treatments. This study assesses the susceptibility of a panel of clinically used photosensitizers to be transported by ABC transporters in vitro. Methods: The involvement of P-glycoprotein (P-gp/ABCB1), breast cancer resistance protein (BCRP/ABCG2), and multidrug resistance-associated protein 1 (MRP1/ABCC1) in the transport of 7 clinically utilized photosensitizers [benzoporphyrin derivative (BPD), temoporfin, redaporfin, talaporfin sodium, rose bengal, methylene blue, and indocyanine green] were investigated using human breast cancer cell lines following well-established protocols. Briefly, parental MCF-7 cells and sublines that overexpress P-gp (MCF-7 TX400), ABCG2 (MCF-7 MX100), or MRP1 (MCF-7/VP) were treated with photosensitizers with and without ABC transporter inhibitors. Intracellular levels of photosensitizers were measured using extraction method and flow cytometry to determine whether the ABC transporters are associated with efflux or uptake of photosensitizers. Results: The ABCG2 inhibitor (fumitremorgin C) and P-gp inhibitor (valspodar) effectively blocked the transport mediated by ABCG2 and P-gp of rose bengal and BPD. Redaporfin showed increased accumulation in the presence of valspodar with flow cytometry. Interestingly, MCF-7/VP cells were found to have reduced intracellular accumulation of rose bengal, which was restored with MRP1 inhibitor (MK571). The cell viability assay showed photodynamic therapy (PDT) resistance with Redaporfin in P-gp-overexpressing cells, BPD in ABCG2- and P-gp-overexpressing cells, and with Rose bengal in ABCG2-, P-gp- and MRP1-overexpressing cells, respectively. However, no change in intracellular retention was observed for other photosensitizers. Conclusion: In summary, our study provided new knowledge that temoporfin, talaporfin sodium, methylene blue, and indocyanine green are not substrates of ABCG2, P-gp, or MRP1. Redaporfin is a substrate for P-gp. BPD is a known substrate of ABCG2 and P-gp. Rose bengal is a substrate of ABCG2, P-gp, and MRP1. The results presented here indicate ABC transporter substrate status as a possible cause for cellular resistance to photodynamic therapy with rose bengal, redaporfin, and BPD.

Objective: Employ a combination of in vivo, cellular-resolution fluorescence microendoscopy (FME) and hyperspectral fluorescence small animal imaging to visualize and quantify photosensitizer delivery to tumor tissues in a murine model of disseminated ovarian cancer. Rationale: Ovarian cancer is the fifth most lethal cancer among women in the United States, accounting for over 20,000 deaths annually. While most patients respond initially to systemic chemotherapy, they often develop resistance to standard platinum regimens. Photodynamic therapy (PDT) is a photochemistry based modality that our group has demonstrated is capable of reversing platinum resistance in human ovarian cancer cells and has shown promise in a murine model of ovarian cancer. An intrinsic advantage to PDT is that the photosensitizers (PS) emit fluorescence and can therefore be used as both an imaging agent and a light-activated therapeutic agent. Effective drug delivery to malignant tissues continues to be a challenge when treating many diseases including ovarian cancer. While the liposomal formulation of benzoporphyrin derivative (BPD) preferentially accumulates in malignant tissues, our group has strived to improve tumor selective delivery of BPD, in order to minimize collateral damage when activating the photosensitizer in vivo, and to increase the BPD tumor payload to enhance tumor cell killing. In order to address these issues we have designed 1) a photoimmunoconjugate (PIC) generated by coupling the PS to the epidermal growth factor receptor (EGFR)-targeting monoclonal antibody Cetuximab; and 2) a “nanocell” construct in which BPD is non-covalently trapped inside polymer nanoparticles, which are then encapsulated inside liposomes. Methods: In the present study we have used PIC- and nanotechnology-based drug delivery platform to improve the tumor selectivity and the delivered payload efficiency of BPD. Real-time assessment of BPD delivery was assessed using two separate imaging modalities. We employed a minimally invasive FME to detect BPD in tumor and normal tissues in situ. Quantitative measurements of BPD fluorescent signals were taken using a hyperspectral small animal imaging system. The presence of tumor and PS localization was confirmed by histology and immunofluorescence. Results and Conclusions: Quantitative imaging demonstrates enhanced BPD delivery by its encapsulation into a nanocell construct. Separately, our imaging results show enhanced tumor selectivity achieved by conjugating BPD to Cetuximab, in comparison to the traditional administration of BPD. The use of the advanced imaging platforms provides insight (and real-time feedback) regarding the localization and concentration of the photosensitizer in tumor tissues, which will lead to more effective treatment planning regimens for ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4320.

Ovarian cancer is the most lethal gynecologic malignancy in women with an increasing number of cases worldwide. Chemoresistance is the main obstacle for ovarian cancer treatment during clinical therapy. Previous studies found that programmed cell death 1 ligand 1 (PD-L1) was associated with chemoresistance of cancer. However, there were little reports about the function of PD-L1 involved in chemoresistance of ovarian cancer. In our study, cisplatin (DDP)-resistant SKOV3 and A2780 ovarian cancer cell lines (SKOV3/DDP and A2780/DDP) were established. We found that the expression of PD-L1 was increased and miR-34a-5p was decreased in DDP-resistant cells. PD-L1 silencing inhibited chemoresistance of DDP-resistant ovarian cancer cells to DDP, as evidenced by decreased proliferation, G1-phase cell cycle arrest and increased apoptosis. Western blot assay showed that in the presence of DDP, PD-L1 silencing decreased multidrug resistance protein 1 and Cyclin D1 protein levels, whereas increased cleaved-caspase-3 and cleaved-PARP protein levels in these cells. Moreover, we demonstrated that miR-34a-5p negatively regulated the expression of PD-L1 by targeting its 3'-untranslated region. The effects of miR-34a-5p mimic on DDP-treated SKOV3/DDP cells were reversed by the overexpression of PD-L1. Moreover, the tumorigenicity of DDP-resistant ovarian cancer cells in nude mice treated with DDP was attenuated by miR-34a-5p in vivo. The combined data indicate that miR-34a-5p/PD-L1 axis regulates DDP chemoresistance of ovarian cancer cells, providing a deeper insight into the treatment for ovarian cancer.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) play an important role in cancer development and progression. However, the mechanism by which they enhance the chemoresistance of ovarian cancer is unknown. Conditioned media of BM-MSCs (BM-MSC-CM) were analyzed using a technique based on microRNA arrays. The most highly expressed microRNAs were selected for testing their effects on glycolysis and chemoresistance in SKOV3 and COC1 ovarian cancer cells. The targeted gene and related signaling pathway were investigated using in silico analysis and in vitro cancer cell models. Kaplan-Merier survival analysis was performed on a population of 59 patients enrolled to analyze the clinical significance of microRNA findings in the prognosis of ovarian cancer. MiR-1180 was the most abundant microRNA detected in BM-MSC-CM, which simultaneously induces glycolysis and chemoresistance (against cisplatin) in ovarian cancer cells. The secreted frizzled-related protein 1 (SFRP1) gene was identified as a major target of miR-1180. The overexpression of miR-1180 led to the activation of Wnt signaling and its downstream components, namely Wnt5a, β-catenin, c-Myc, and CyclinD1, which are responsible for glycolysis-induced chemoresistance. The miR-1180 level was inversely correlated with SFRP1 mRNA expression in ovarian cancer tissue. The overexpressed miR-1180 was associated with a poor prognosis for the long-term (96-month) survival of ovarian cancer patients. BM-MSCs enhance the chemoresistance of ovarian cancer by releasing miR-1180. The released miR-1180 activates the Wnt signaling pathway in cancer cells by targeting SFRP1. The enhanced Wnt signaling upregulates the glycolytic level (i.e. Warburg effect), which reinforces the chemoresistance property of ovarian cancer cells.

The immunological response against cancer is a critical balance between immune-activating and immune-suppressing mechanisms. Ovarian cancer creates a suppressive microenvironment to escape immune elimination; however, the molecular mechanisms are poorly understood, and it is unclear whether chemotherapeutic drugs exert an immunoreactive or immunosuppressive effect on the tumor microenvironment. 4-1BB ligand (4-1BBL/CD157) and OX-40 ligand (OX-40L/CD252) are important regulators of effector cytotoxic T-cells activity. This study demonstrates that expression of positive co-stimulatory molecules, OX-40L and 4-1BBL, is suppressed while expression of immunosuppressive molecule programmed death ligand-1 (PD-L1/CD274) is enhanced in chemoresistant cells compared to parental chemosensitive ovarian cancer cells. Here, the molecular mechanisms of silencing of OX-40L and 4-1BBL expression were investigated in chemoresistant A2780-AD ovarian cancer cells. The suppression of OX-40L and 4-1BBL are due to DNA hypermethylation and histone deacetylation, two important mechanisms that contribute to gene silencing during cancer progression. We identify important epigenetic regulators, histone deacetylase 1/3 (HDAC1/HDAC3) and DNA methyltransferase 1 (DNMT1), that exhibit aberrant association with OX-40L and 4-1BBL promoters in chemoresistant ovarian cancer cells. Knockdown of HDAC1 or DNMT1 expression, and pharmacological inhibition of DNMT or HDAC enzymatic activity, significantly increase OX-40L and 4-1BBL expression in chemoresistant cells. This study suggests that loss of histone acetylation and accumulation of DNA methylation correlates with suppressed expression of OX-40L and 4-1BBL in chemoresistant ovarian cancer cells. This study marks the first report of the regulation of these two molecules by histone deacetylation and DNA methylation in chemoresistant ovarian cancer cells.

BackgroundA critical therapeutic challenge in epithelial ovarian carcinoma is the development of chemoresistance among tumor cells following exposure to first line chemotherapeutics. The molecular and genetic changes that drive the development of chemoresistance are unknown, and this lack of mechanistic insight is a major obstacle in preventing and predicting the occurrence of refractory disease. We have recently shown that Regulators of G-protein Signaling (RGS) proteins negatively regulate signaling by lysophosphatidic acid (LPA), a growth factor elevated in malignant ascites fluid that triggers oncogenic growth and survival signaling in ovarian cancer cells. The goal of this study was to determine the role of RGS protein expression in ovarian cancer chemoresistance.ResultsIn this study, we find that RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant to chemotherapy compared with parental, chemo-sensitive cells in gene expression datasets of multiple models of chemoresistance. Further, exposure of SKOV-3 cells to cytotoxic chemotherapy causes acute, persistent downregulation of RGS10 and RGS17 transcript expression. Direct inhibition of RGS10 or RGS17 expression using siRNA knock-down significantly reduces chemotherapy-induced cell toxicity. The effects of cisplatin, vincristine, and docetaxel are inhibited following RGS10 and RGS17 knock-down in cell viability assays and phosphatidyl serine externalization assays in SKOV-3 cells and MDR-HeyA8 cells. We further show that AKT activation is higher following RGS10 knock-down and RGS 10 and RGS17 overexpression blocked LPA mediated activation of AKT, suggesting that RGS proteins may blunt AKT survival pathways.ConclusionsTaken together, our data suggest that chemotherapy exposure triggers loss of RGS10 and RGS17 expression in ovarian cancer cells, and that loss of expression contributes to the development of chemoresistance, possibly through amplification of endogenous AKT signals. Our results establish RGS10 and RGS17 as novel regulators of cell survival and chemoresistance in ovarian cancer cells and suggest that their reduced expression may be diagnostic of chemoresistance.

ABC transporter gene expression in benign and malignant ovarian tissue

ABCB5 gene amplification in human leukemia cells

Regulator of G-protein signaling 2 (RGS2) is a GTPase-activating protein functioning as an inhibitor of G-protein coupled receptors (GPCRs). RGS2 dysregulation was implicated in solid tumour development and RGS2 downregulation has been reported in prostate and ovarian cancer progression. However, the molecular mechanism by which RGS2 expression is suppressed in ovarian cancer remains unknown. The expression and epigenetic regulation of RGS2 in chemosensitive and chemoresistant ovarian cancer cells were determined by qRT-PCR and chromatin immunoprecipitation assays, respectively. In the present study, the molecular mechanisms contributing to the loss of RGS2 expression were determined in ovarian cancer. The data indicated that suppression of RGS2 gene in chemoresistant ovarian cancer cells, in part, due to accumulation of histone deacetylases (HDACs) and DNA methyltransferase I (DNMT1) at the promoter region of RGS2. Inhibition of HDACs or DNMTs significantly increases RGS2 expression. These results suggest that epigenetic changes in histone modifications and DNA methylation may contribute to the loss of RGS2 expression in chemoresistant ovarian cancer cells. The results further suggest that class I HDACs and DNMT1 contribute to the suppression of RGS2 during acquired chemoresistance and support growing evidence that inhibition of HDACs/DNMTs represents novel therapeutic approaches to overcome ovarian cancer chemoresistance.

Background: Platinum is the most commonly used drug for the treatment of ovarian cancer. However, resistance to platinum has also become a serious clinical problem. The mutation of BRCA (BRCA1/2) in ovarian cancer, is the major cause of the occurrence of tumors. Our previous microarray data showed that expression level of CDC-like kinase (Clk2) was higher in ovarian cancer tissues than the compared normal tissues. As a protein kinase, Clk2 phosphorylates and activates BRCA1. Methods: 1.Quantitative PCR and IHC were conducted to verify the expression of Clk2 in normal ovarian and tumor tissues.2.The WB was used to examine the correlation between Clk2 protein level and the time and concentration of cisplatin, as well as the protein expression of Clk2 in the platinum sensitive and resistant cell strains of ovarian cancer.3.After knocking down Clk2, the IC50 of cisplatin, the DNA damage level and the apoptosis ratio of drug-resistant cell were examined.4.Tumor formation model was made in nude mice to test the tumor size of drug-resistant cell.5.Protein profiling was analysed to screen proteins that bind to endogenous Clk2 in ovarian cancer cells. Co-IP was used to prove the binding of Clk2 to BRCA1 and to detect the relationship between their binding levels and the time and concentration gradient of platinum.6.The effect of Clk2 overexpression on the activation of DNA damage-related proteins was detected by WB.7.IHC was used to observe of changes of p-BRCA1 and formation of DNA damage repair focus after knockout of Clk2.8.The correlation between p-BRCA1 expression and Clk2 in ovarian cancer was demonstrated via IHC.9.The recombinant kinase experiment was conducted to analyze the ability of the Clk2 protein kinase to activate BRCA1.10.The related pathway inhibitors were used to preliminarily screen for the signaling pathways in which cisplatin induced upregulation of Clk2 and validate.11.The "synergistic lethal effect" of the combination of Clk2 with PARP inhibitors was explored preliminarily. Conclusions: 1.Compared with normal ovarian tissues, Clk2 is up-regulated in ovarian cancer tissue, especially in chemo-resistant ovarian cancer tissues, and high expression of Clk2 indicates poor prognosis. 2.Clk2 enhances DNA damage repair and promotes drug resistance in ovarian cancer. 3.Clk2 activates BRCA1 in chemo-resistant ovarian cancer cells. 4.Platinum activates p38 signal, which contributes to the phosphorylation of T343 in Clk2 Activation Loop, then Clk2 protein is stabilized. And Clk2 can activate BRCA1 in chemo-resistant ovarian cancer cells. 5.A preliminary study shows that combining Clk2 and PARP inhibitors can form a "synergistic lethal effect" and increase DNA damage in ovarian cancer platinum-resistant cells. Citation Format: Shuting Huang, Yinan Jiang, Min Zheng. The functional mechanism of Clk2 supporting the development of chemoresistance in non-BRCA1 deficient ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4088.

Chemo-sensitive and chemo-resistant ovarian cancer cells show differences in cellular processes leading to pyroptotic cell death.

RGS10 is an important regulator of cell survival and chemoresistance in ovarian cancer. We recently showed that RGS10 transcript expression is suppressed during acquired chemoresistance in ovarian cancer. The suppression of RGS10 is due to DNA hypermethylation and histone deacetylation, two important mechanisms that contribute to silencing of tumor suppressor genes during cancer progression. Here, we fully investigate the molecular mechanisms of epigenetic silencing of RGS10 expression in chemoresistant A2780-AD ovarian cancer cells. We identify two important epigenetic regulators, HDAC1 and DNMT1, that exhibit aberrant association with RGS10 promoters in chemoresistant ovarian cancer cells. Knockdown of HDAC1 or DNMT1 expression, and pharmacological inhibition of DNMT or HDAC enzymatic activity, significantly increases RGS10 expression and cisplatin-mediated cell death. Finally, DNMT1 knock down also decreases HDAC1 binding to the RGS10 promoter in chemoresistant cells, suggesting HDAC1 recruitment to RGS10 promoters requires DNMT1 activity. Our results suggest that HDAC1 and DNMT1 contribute to the suppression of RGS10 during acquired chemoresistance and support inhibition of HDAC1 and DNMT1 as an adjuvant therapeutic approach to overcome ovarian cancer chemoresistance.

Ovarian cancer is one of the most lethal gynaecologic malignancies. This can be attributed to the emergence of multidrug resistance in the clinic due to the over-expression of drug-efflux pumps such as P-gp. To overcome this, we have developed a liposomal platform for the the co-delivery of potent P-gp inhibitors with chemotherapeutic drugs. Purpose: To allow for the effective reversal of chemo-resistance in ovarian cancer cells using liposomes co-loaded with tariquidar and paclitaxel. Methods: PEGylated liposomes composed of eggphosphatidylcholine, cholesterol and DOTAP loaded with equimolar amounts of tariquidar (XR) and paclitaxel (PCT) were prepared by the thin film hydration method followed by extrusion. Evaluation of drug resistance of ovarian cancer lines SKOV3, HeyA8 and Tynku was carried out by quantifying MDR1 and MRP1 receptor expression followed by a rhodamine123 exclusion assay. The cytotoxicity of the formulations on the above cancer cell lines was evaluated using the Promega cell viability assay. The effects of the formulations were evaluated in fluorescently stained cells using the iCyte® imaging cytometer (Thorlabs, USA). Long-term kinetic analysis of unstained live cultures was perfomed by holographic imaging cytometer Holomonitor ® M4 (Phase Holographic Imaging, Sweden) and fluorescence microscopy. Results: The liposomes had a particle size distribution of about 200 nm with a zeta potential of 30 mV. As compared to MRP1, a 2.4 fold, 1.9-fold and 1.6-fold higher expression of MDR1 was seen in the HeyA8-MDR, SKOV3-TR and Tynku-R cells respectively over their drug-sensitive counterparts. Using the rhodamine exclusion assay, 14 nM of liposomal XR showed better rhodamine inclusion than 140 nM free XR. In vitro cytotoxicity experiments showed that the drug co-loaded formulation was able to effectively overcome resistance to PCT even at low PCT doses. Using live-cell imaging, paclitaxel-induced arrest of cell cycle progression and onset of apoptosis following this mitotic catastrophe was visible from 1.5 μM to 50 nM only with the co-loaded liposomes. The cell cycle arrest was further visualized using time-lapse Holomonitor M4 which showed the increasing incidence of cells arrested in mitosis over time. Using fluorescence microscopy, we were able to clearly visualize the presence of polymerized microtubules around the cell nucleus as a result of the paclitaxel-mediated microtubule dysfunction. Conclusions: Uniform liposomes co-loaded with tariquidar and paclitaxel were prepared and characterized. The co-loaded liposomes were able to effectively reverse chemoresistance and induce cytotoxicity in a variety of drug-resistant ovarian cancers. The formulations were shown to arrest cell cycle, preventing its progression to the G1 phase. This arrest of cell cycle was shown to be due to the paclitaxel-mediated micro-tubule dysfunction. Note: This abstract was not presented at the meeting. Citation Format: Shravan K. Sriraman, Yilin Zhang, Ed Luther, Ernst Lengyel, Vladimir Torchilin, Vladimir Torchilin. Reversal of chemoresistance in ovarian cancer cells by the liposomal co-delivery of MDR inhibitors and paclitaxel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4416. doi:10.1158/1538-7445.AM2015-4416

Chemoresistance is a major therapeutic problem and the current knowledge on cellular mechanisms involved is incomplete. In the present study, we have investigated the possible involvement of Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP) in ovarian cancer resistance by comparing chemosensitive (OV2008) and chemoresistant (C13*) ovarian cancer cells treated with cisplatin in vitro, and/or transfected with FLIP sense cDNA or FLIP small interfering RNA (siRNA) and determining FLIP protein content, cleavage of caspase-8 and caspase-3 and apoptosis. Cisplatin significantly decreased FLIP protein level, induced cleavage of caspase-8 and caspase-3 and apoptosis in a concentration-dependent manner in cisplatin-sensitive but not -resistant cells. While overexpression of FLIP-attenuated cisplatin-induced cleavage of caspase-8 and caspase-3 and apoptosis in chemosensitive cells, downregulation of FLIP in chemoresistant cells by siRNA increased apoptosis induced by cisplatin. These results suggest that FLIP plays a significant role in the regulation of apoptosis in human ovarian cancer cells and their sensitivity to cisplatin. This cell survival factor may be an important determinant in chemoresistance in ovarian cancer and may serve as a molecular target for the development of novel therapy for chemoresistant ovarian cancer.

ABC transporters as mediators of drug resistance and contributors to cancer cell biology.