Sung-Han Hsiao

Overexpression of ATP-binding cassette transporter ABCG2 as a potential mechanism of acquired resistance to vemurafenib in BRAF(V600E) mutant cancer cells.

Melanoma is the most serious type of skin cancer with a high potential for metastasis and very low survival rates. The discovery of constitutive activation of the BRAF kinase caused by activating BRAF(V600E) kinase mutation in most melanoma patients led to the discovery of the first potent BRAF(V600E) signaling inhibitor, vemurafenib. Vemurafenib was effective in treating advanced melanoma patients and was proposed for the treatment of other BRAF(V600E) mutant cancers as well. Unfortunately, the success of vemurafenib was hampered by the rapid development of acquired resistance in different types of BRAF(V600E) mutant cancer cells. It becomes important to identify and evaluate all of the potential mechanisms of cellular resistance to vemurafenib. In this study, we characterized the interactions of vemurafenib with three major ATP-binding cassette (ABC) transporters, ABCB1, ABCC1 and ABCG2. We found that vemurafenib stimulated the ATPase activity and potently inhibited drug efflux mediated by ABCB1 and ABCG2. Vemurafenib also restored drug sensitivity in ABCG2-overexpressing cells. Moreover, we revealed that in the presence of functional ABCG2, BRAF kinase inhibition by vemurafenib is reduced in BRAF(V600E) mutant A375 cells. Taken together, our findings indicate that ABCG2 confers resistance to vemurafenib in A375 cells, suggesting involvement of this transporter in acquired resistance to vemurafenib. Thus, combination chemotherapy targeting multiple pathways could be an effective therapeutic strategy to overcome acquired resistance to vemurafenib for cancers harboring the BRAF(V600E) mutation.

Human ABCB1 (P-glycoprotein) and ABCG2 mediate resistance to BI 2536, a potent and selective inhibitor of Polo-like kinase 1.

The overexpression of the serine/threonine specific Polo-like kinase 1 (Plk1) has been detected in various types of cancer, and thus has fast become an attractive therapeutic target for cancer therapy. BI 2536 is the first selective inhibitor of Plk1 that inhibits cancer cell proliferation by promoting G2/M cell cycle arrest at nanomolar concentrations. Unfortunately, alike most chemotherapeutic agents, the development of acquired resistance to BI 2536 is prone to present a significant therapeutic challenge. One of the most common mechanisms for acquired resistance in cancer chemotherapy is associated with the overexpression of ATP-binding cassette (ABC) transporters ABCB1, ABCC1 and ABCG2. Here, we discovered that overexpressing of either ABCB1 or ABCG2 is a novel mechanism of acquired resistance to BI 2536 in human cancer cells. Moreover, BI 2536 stimulates the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner, and inhibits the drug substrate transport mediated by these transporters. More significantly, the reduced chemosensitivity and BI 2536-mediated G2/M cell cycle arrest in cancer cells overexpressing either ABCB1 or ABCG2 can be significantly restored in the presence of selective inhibitor or other chemotherapeutic agents that also interact with ABCB1 and ABCG2, such as tyrosine kinase inhibitors nilotinib and lapatinib. Taken together, our findings indicate that in order to circumvent ABCB1 or ABCG2-mediated acquired resistance to BI 2536, a combined regimen of BI 2536 and inhibitors or clinically active drugs that potently inhibit the function of ABC drug transporters, should be considered as a potential treatment strategy in the clinic.

Alpha-mangostin reverses multidrug resistance by attenuating the function of the multidrug resistance-linked ABCG2 transporter

The ATP-binding cassette (ABC) drug transporter ABCG2 can actively efflux a wide variety of chemotherapeutic agents out of cancer cells and subsequently reduce the intracellular accumulation of these drugs. Therefore, the overexpression of ABCG2 often contributes to the development of multidrug resistance (MDR) in cancer cells, which is one of the major obstacles to successful cancer chemotherapy. Moreover, ABCG2 is highly expressed in various tissues including the intestine and blood-brain barrier (BBB), limiting the absorption and bioavailability of many therapeutic agents. For decades, the task of developing a highly effective synthetic inhibitor of ABCG2 has been hindered mostly by the intrinsic toxicity, the lack of specificity, and complex pharmacokinetics. Alternatively, considering the wide range of diversity and relatively nontoxic nature of natural products, developing potential modulators of ABCG2 from natural sources is particularly valuable. α-Mangostin is a natural xanthone derived from the pericarps of mangosteen (Garcinia mangostana L.) with various pharmacological purposes, including suppressing angiogenesis and inducing cancer cell growth arrest. In this study, we demonstrated that at nontoxic concentrations, α-mangostin effectively and selectively inhibits ABCG2-mediated drug transport and reverses MDR in ABCG2-overexpressing MDR cancer cells. Direct interactions between α-mangostin and the ABCG2 drug-binding site(s) were confirmed by stimulation of ATPase activity and by inhibition of photolabeling of the substrate-binding site(s) of ABCG2 with [125I]iodoarylazidoprazosin. In summary, our findings show that α-mangostin has great potential to be further developed into a promising modulator of ABCG2 for reversing MDR and for its use in combination therapy for patients with MDR tumors.

The positive inotropic agent DPI-201106 selectively reverses ABCB1-mediated multidrug resistance in cancer cell lines

The overexpression of ABCB1 in cancer cells is a major factor contributing to the development of multidrug resistance (MDR) and treatment failure in cancer patients. Therefore, re-sensitization of MDR cancer cells to anticancer drugs remains an important aspect in chemotherapy. The progress in developing clinically applicable synthetic inhibitors of ABCB1 has been slow, mostly due to complications associated with intrinsic toxicities and unforeseen drug-drug interactions. Here, we explored the drug-repositioning approach for cancer therapy by targeting ABCB1-mediated MDR in human cancer cells. We found that DPI-201106, a positive inotropic agent, selectively inhibits the drug efflux function of ABCB1, and in doing so, re-sensitizes ABCB1-overexpressing MDR cancer cells to conventional anticancer drugs. Furthermore, the ATPase activity of ABCB1 and docking analysis of DPI-201106 in the drug-binding pocket of ABCB1 were determined to confirm the interaction between DPI-201106 and ABCB1 protein. In summary, we revealed an additional action and a potential clinical application of DPI-201106 to reverse ABCB1-mediated MDR in human cancer cells, which may be beneficial for cancer patients who have developed multidrug resistance and no longer respond to conventional chemotherapy, and should be further investigated.

SIS3, a specific inhibitor of Smad3 reverses ABCB1- and ABCG2-mediated multidrug resistance in cancer cell lines

One of the major challenges in cancer chemotherapy is the development of multidrug resistance phenomenon attributed to the overexpression of ATP-binding cassette (ABC) transporter ABCB1 or ABCG2 in cancer cells. Therefore, re-sensitizing MDR cancer cells to chemotherapy by directly inhibiting the activity of ABC transporters has clinical relevance. Unfortunately, previous attempts of developing clinically applicable synthetic inhibitors have failed, mostly due to problems associated with toxicity and unforeseen drug-drug interactions. An alternative approach is by repositioning drugs with known pharmacological properties as modulators of ABCB1 and ABCG2. In this study, we discovered that the transport function of ABCB1 and ABCG2 is strongly inhibited by SIS3, a specific inhibitor of Smad3. More importantly, SIS3 enhances drug-induced apoptosis and resensitizes ABCB1- and ABCG2-overexpressing cancer cells to chemotherapeutic drugs at non-toxic concentrations. These findings are further supported by ATPase assays and by a docking analysis of SIS3 in the drug-binding pockets of ABCB1 and ABCG2. In summary, we revealed an additional action of SIS3 that re-sensitizes MDR cancer cells and a combination therapy with this drug and other chemotherapeutic agents may be beneficial for patients with MDR tumors.

Human ATP-binding cassette transporters ABCB1 and ABCG2 confer resistance to histone deacetylase 6 inhibitor ricolinostat (ACY-1215) in cancer cell lines

Graphical abstract Figure. No caption available. &NA; Ricolinostat is the first orally available, selective inhibitor of histone deacetylase 6 (HDAC6), currently under evaluation in clinical trials in patients with various malignancies. It is likely that the inevitable emergence of resistance to ricolinostat is likely to reduce its clinical effectiveness in cancer patients. In this study, we investigated the potential impact of multidrug resistance‐linked ATP‐binding cassette (ABC) transporters ABCB1 and ABCG2 on the efficacy of ricolinostat, which may present a major hurdle to its development as an anticancer drug in the future. We demonstrated that the overexpression of ABCB1 or ABCG2 reduces the intracellular accumulation of ricolinostat, resulting in reduced efficacy of ricolinostat to inhibit the activity of HDAC6 in cancer cells. Moreover, the efficacy of ricolinostat can be fully restored by inhibiting the drug efflux function of ABCB1 and ABCG2 in drug‐resistant cancer cells. In conclusion, our results provide some insights into the basis for the development of resistance to ricolinostat and suggest that co‐administration of ricolinostat with a modulator of ABCB1 or ABCG2 could overcome ricolinostat resistance in human cancer cells, which may be relevant to its use in the clinic.

Overexpression of ATP-Binding Cassette Subfamily G Member 2 Confers Resistance to Phosphatidylinositol 3-Kinase Inhibitor PF-4989216 in Cancer Cells

Deregulated activation of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently found in human cancers, which plays a key role in promoting cancer proliferation and resistance to anticancer therapies. Therefore, developing inhibitors targeting key components of the PI3K/Akt/mTOR signaling pathway has great clinical significance. PF-4989216 is a novel, orally available small-molecule drug that was developed to selectively inhibit the PI3K/Akt/mTOR signaling pathway and subsequent cancer cell proliferation. PF-4989216 exhibited potent and selective inhibition against PI3K kinase activity in preclinical small-cell lung cancer (SCLC) models, and was especially effective against the proliferation of SCLCs harboring PIK3CA mutation. Unfortunately, in addition to innate resistance mechanisms, drug extrusion by the efflux pumps may also contribute to the development of acquired resistance to PI3K inhibitors in cancer cells. The overexpression of ATP-binding cassette (ABC) drug transporters ABCB1 and ABCG2 is one of the most common mechanisms for reducing intracellular drug concentration and developing multidrug resistance, which remains a substantial challenge to the effective treatment of cancer. In this study, we report the discovery of ABCG2 overexpression as a mechanism of resistance to PI3K inhibitor PF-4989216 in human cancer cells. We demonstrated that the inhibition of Akt and downstream S6RP phosphorylation by PF-4989216 were significantly reduced in ABCG2-overexpressing human cancer cells. Moreover, overexpression of ABCG2 in various cancer cell lines confers significant resistance to PF-4989216, which can be reversed by an inhibitor or competitive substrate of ABCG2, indicating that ABCG2-mediated transport alone can sufficiently reduce the intracellular concentration of PF-4989216.

The Overexpression of ABCG2 Reduces the Efficacy of Volasertib (BI 6727) and GSK641364 in Human S1-M1-80 Colon Carcinoma Cells

The polo-like kinase 1 (Plk1) is one of the key regulators in cell cycle progression. Plk1 is overexpressed in many types of cancer and promotes the proliferation of cancer cells. Inhibition of Plk1 activity induces G2/M cell cycle arrest and reduces cancer cell viability. Volasertib and GSK461364 are selective inhibitors of Plk1, active against a wide range of tumor cells at nanomolar concentrations. In this study, while examining the effectiveness of Plk1 inhibitors against multiple human colon cancer cell lines, we discovered that the overexpression of ATP-binding cassette (ABC) drug transporter ABCG2 in human S1-M1-80 colon cancer cells confers resistance to volasertib and GSK461364. Moreover, we found that ABCG2-transfected HEK293 cells were also resistant to both Plk1 inhibitors. We revealed that volasertib and GSK461364 inhibited the function of ABCG2 in a concentration dependent manner, and had no significant effect on the protein expression of ABCG2. More importantly, we showed that the G2/M cell cycle arrest induced by volasertib or GSK461364 was significantly reduced in S1-M1-80 cells, and that ABCG2-mediated drug resistance to Plk1 inhibitors can be restored by inhibition of ABCG2 function. Therefore, the development of ABCG2-mediated drug resistance to volasertib and GSK461364 in cancer clearly present a significant therapeutic challenge, and a better treatment strategy should be further investigated.

Abstract 3398: Overexpression of ATP-binding cassette transporter ABCG2 as a potential mechanism of acquired resistance to vemurafenib in BRAF(V600E) mutant cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnMelanoma is the most serious type of skin cancer with a high potential for metastasis and very low survival rates. The discovery of constitutive activation of the BRAF kinase caused by activating BRAF(V600E) kinase mutation in most melanoma patients led to the discovery of the first potent BRAF(V600E) signaling inhibitor, vemurafenib. Vemurafenib was effective in treating advanced melanoma patients and was proposed for the treatment of other BRAF(V600E) mutant cancers as well. Unfortunately, the success of vemurafenib was hampered by the rapid development of acquired resistance in different types of BRAF(V600E) mutant cancer cells. It becomes important to identify and evaluate all of the potential mechanisms of cellular resistance to vemurafenib. In this study, we characterized the interactions of vemurafenib with three major ATP-binding cassette (ABC) transporters, ABCB1, ABCC1 and ABCG2. We found that vemurafenib stimulated the ATPase activity and potently inhibited drug efflux mediated by ABCB1 and ABCG2. Vemurafenib also restored drug sensitivity in ABCG2-overexpressing cells. Moreover, we revealed that in the presence of functional ABCG2, BRAF kinase inhibition by vemurafenib is reduced in BRAF(V600E) mutant A375 cells. Taken together, our findings indicate that ABCG2 confers resistance to vemurafenib in A375 cells, suggesting involvement of this transporter in acquired resistance to vemurafenib. Thus, combination chemotherapy targeting multiple pathways could be an effective therapeutic strategy to overcome acquired resistance to vemurafenib for cancers harboring the BRAF(V600E) mutation.nnCitation Format: Chung-Pu Wu, Hong-May Sim, Yang-Hui Huang, Yen-Chen Liu, Sung-Han Hsiao, Hsing-Wen Cheng, Yan-Qing Li, Suresh Ambudkar, Sheng-Chieh Hsu. Overexpression of ATP-binding cassette transporter ABCG2 as a potential mechanism of acquired resistance to vemurafenib in BRAF(V600E) mutant cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3398. doi:10.1158/1538-7445.AM2013-3398