Coexpression of ABCB1 and ABCG2 in a Cell Line Model Reveals Both Independent and Additive Transporter Function.

Although overexpression of multiple ATP-binding cassette transporters has been reported in clinical samples, few studies have examined how coexpression of multiple transporters affected resistance to chemotherapeutic drugs. We therefore examined how coexpression of ABCB1 (P-glycoprotein) and ABCG2 contributes to drug resistance in a cell line model. HEK293 cells were transfected with vector-encoding full-length ABCB1, ABCG2, or a bicistronic vector containing both genes, each under the control of a separate promoter. Cells transfected with both transporters (B1/G2 cells) demonstrated high levels of both transporters, and uptake of both the ABCB1-specific substrate rhodamine 123 and the ABCG2-specific substrate pheophorbide a was reduced when examined by flow cytometry. B1/G2 cells were also cross-resistant to the ABCB1 substrate doxorubicin, the ABCG2 substrate topotecan, as well as mitoxantrone and the cell cycle checkpoint kinase 1 inhibitor prexasertib, both of which were found to be substrates of both ABCB1 and ABCG2. When B1/G2 cells were incubated with both rhodamine 123 and pheophorbide a, transport of both compounds was observed, suggesting that ABCB1 and ABCG2, when coexpressed, can function independently to transport substrates. ABCB1 and ABCG2 also functioned additively to transport the common fluorescent substrates mitoxantrone and BODIPY-prazosin, as it was necessary to inhibit both transporters to prevent efflux from B1/G2 cells. ABCG2 expression was also found to decrease the efficacy of the ABCB1 inhibitor tariquidar in B1/G2 cells. Thus, ABCB1 and ABCG2 can independently and additively confer resistance to substrates, underscoring the need to inhibit multiple transporters when they are coexpressed.

Targeting mitochondrial hexokinases increases efficacy of histone deacetylase inhibitors in solid tumor models.

Hexokinase 1 and 2 have been shown to inhibit Bak- and Bax-mediated apoptosis, leading us to combine the histone deacetylase inhibitor romidepsin with clotrimazole or bifonazole, two compounds that reportedly decrease mitochondrial localization of hexokinases. Cancer cell lines derived from breast, kidney, lung, colon or ovarian cancers were treated with a short-term exposure to 25 ng/ml romidepsin combined with either clotrimazole or bifonazole. The combination of romidepsin with 25 µM clotrimazole or bifonazole resulted in increased annexin staining compared to cells treated with any of the drugs alone. Cell death was caspase-mediated, as the pan-caspase inhibitor Q-VD-OPh was found to inhibit apoptosis induced by the combination. A549 lung cancer cells or HCT-116 cells deficient in Bak and Bax were also resistant to apoptosis with the combination implicating the intrinsic apoptotic pathway. We found that a 24 h treatment with clotrimazole or bifonazole decreased total hexokinase 2 expression, resulting in a 76% or 60% decrease, respectively, of mitochondrial expression of hexokinase 2. Mitochondrial hexokinase 1 levels increased 2-fold or less. Our work suggests that the combination of a short-term romidepsin treatment with bifonazole or clotrimazole leads to increased apoptosis, most likely due to decreased mitochondrial expression of hexokinase 2.

Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells.

Glycogen synthase kinase-3 (GSK-3) activity regulates multiple signal transduction pathways and is also a key component of the network responsible for maintaining stem cell pluripotency. Genetic deletion of Gsk-3α and Gsk-3ß or inhibition of GSK-3 activity via small molecules promotes stem cell pluripotency, yet the mechanism underlying the role for GSK-3 in this process remains ambiguous. Another cellular process that has been shown to affect stem cell pluripotency is mRNA methylation (m6A). Here, we describe an intersection between these components, the regulation of m6A by GSK-3. We find that protein levels for the RNA demethylase, FTO (fat mass and obesity-associated protein), are elevated in Gsk-3α;Gsk-3ß-deficient mouse embryonic stem cells (ESCs). FTO is normally phosphorylated by GSK-3, and MS identified the sites on FTO that are phosphorylated in a GSK-3-dependent fashion. GSK-3 phosphorylation of FTO leads to polyubiquitination, but in Gsk-3 knockout ESCs, that process is impaired, resulting in elevated levels of FTO protein. As a consequence of altered FTO protein levels, mRNAs in Gsk-3 knockout ESCs have 50% less m6A than WT ESCs, and m6A-Seq analysis reveals the specific mRNAs that have reduced m6A modifications. Taken together, we provide the first evidence for how m6A demethylation is regulated in mammalian cells and identify a putative novel mechanism by which GSK-3 activity regulates stem cell pluripotency.

Blocking downstream signaling pathways in the context of HDAC inhibition promotes apoptosis preferentially in cells harboring mutant Ras.

We previously demonstrated activation of the mitogen-activated protein kinase (MAPK) pathway in a series of romidepsin-selected T-cell lymphoma cell lines as a mechanism of resistance to the histone deacetylase inhibitor (HDI), romidepsin. As Ras mutation leads to activation of both the MAPK and the phosphoinositide 3-kinase (PI3K) pathway, we examined whether combining romidepsin with small molecule pathway inhibitors would lead to increased apoptosis in cancers harboring Ras mutations. We treated 18 Ras mutant or wild-type cell lines with romidepsin in the presence of a MEK inhibitor (PD-0325901) and/or an AKT inhibitor (MK-2206) and examined apoptosis by flow cytometry. A short-term treatment schedule of romidepsin (25 ng/ml for 6 h) was used to more closely model clinical administration. Romidepsin in combination with a MEK and an AKT inhibitor induced apoptosis preferentially in cells harboring mutant versus wild-type Ras (69.1% vs. 21.1%, p < 0.0001). Similar results were found in a subset of cell lines when belinostat was combined with the MEK and AKT inhibitors and when romidepsin was combined with the dual extracellular signaling-related kinase (ERK)/PI3K inhibitor, D-87503, which inhibited both the MAPK and PI3K pathways at 5-10 µM. The observed apoptosis was caspase-dependent and required Bak and Bax expression. Cells with wild-type or mutant Ras treated with romidepsin alone or in combination with the MEK inhibitor displayed increased expression of proapoptotic Bim. We thus conclude that cancers bearing Ras mutations, such as pancreatic cancer, can be targeted by the combination of an HDI and a dual inhibitor of the MAPK and PI3K pathways.