Agnes Basseville

Targeting MDR in breast and lung cancer: Discriminating its potential importance from the failure of drug resistance reversal studies

This special issue of Drug Resistance Updates is dedicated to multidrug resistance protein 1 (MDR-1), 35 years after its discovery. While enormous progress has been made and our understanding of drug resistance has become more sophisticated and nuanced, after 35 years the role of MDR-1 in clinical oncology remains a work in progress. Despite clear in vitro evidence that P-glycoprotein (Pgp), encoded by MDR-1, is able to dramatically reduce drug concentrations in cultured cells, and that drug accumulation can be increased by small molecule inhibitors, clinical trials testing this paradigm have mostly failed. Some have argued that it is no longer worthy of study. However, repeated analyses have demonstrated MDR-1 expression in a tumor is a poor prognostic indicator leading some to conclude MDR-1 is a marker of a more aggressive phenotype, rather than a mechanism of drug resistance. In this review we will re-evaluate the MDR-1 story in light of our new understanding of molecular targeted therapy, using breast and lung cancer as examples. In the end we will reconcile the data available and the knowledge gained in support of a thesis that we understand far more than we realize, and that we can use this knowledge to improve future therapies.

Histone Deacetylase Inhibitors: Emerging Mechanisms of Resistance

The histone deacetylase inhibitors (HDIs) have shown promise in the treatment of a number of hematologic malignancies, leading to the approval of vorinostat and romidepsin for the treatment of cutaneous T-cell lymphoma and romidepsin for the treatment of peripheral T-cell lymphoma by the U.S. Food and Drug Administration. Despite these promising results, clinical trials with the HDIs in solid tumors have not met with success. Examining mechanisms of resistance to HDIs may lead to strategies that increase their therapeutic potential in solid tumors. However, relatively few examples of drug-selected cell lines exist, and mechanisms of resistance have not been studied in depth. Very few clinical translational studies have evaluated resistance mechanisms. In the current review, we summarize many of the purported mechanisms of action of the HDIs in clinical trials and examine some of the emerging resistance mechanisms.

Irinotecan induces steroid and xenobiotic receptor (SXR) signaling to detoxification pathway in colon cancer cells

BackgroundResistance to chemotherapy remains one of the principle obstacles to the treatment of colon cancer. In order to identify the molecular mechanism of this resistance, we investigated the role of the steroid and xenobiotic receptor (SXR) in the induction of drug resistance. Indeed, this nuclear receptor plays an important role in response to xenobiotics through the upregulation of detoxification genes. Following drug treatments, SXR is activated and interacts with the retinoid X receptor (RXR) to induce expression of some genes involved in drug metabolism such as phase I enzyme (like CYP), phase II enzymes (like UGT) and transporters (e.g. MDR1).ResultsIn this study, we have shown that endogenous SXR is activated in response to SN-38, the active metabolite of the anticancer drug irinotecan, in human colon cancer cell lines. We have found that endogenous SXR translocates into the nucleus and associates with RXR upon SN-38 treatment. Using ChIP, we have demonstrated that endogenous SXR, following its activation, binds to the native promoter of the CYP3A4 gene to induce its expression. RNA interference experiments confirmed SXR involvement in CYP3A4 overexpression and permitted us to identify CYP3A5 and MRP2 transporter as SXR target genes. As a consequence, cells overexpressing SXR were found to be less sensitive to irinotecan treatment.ConclusionsAltogether, these results suggest that the SXR pathway is involved in colon cancer irinotecan resistance in colon cancer cell line via the upregulation of select detoxification genes.

Histone deacetylase inhibitors influence chemotherapy transport by modulating expression and trafficking of a common polymorphic variant of the ABCG2 efflux transporter

Histone deacetylase inhibitors (HDI) have exhibited some efficacy in clinical trials, but it is clear that their most effective applications have yet to be fully determined. In this study, we show that HDIs influence the expression of a common polymorphic variant of the chemotherapy drug efflux transporter ABCG2, which contributes to normal tissue protection. As one of the most frequent variants in human ABCG2, the polymorphism Q141K impairs expression, localization, and function, thereby reducing drug clearance and increasing chemotherapy toxicity. Mechanistic investigations revealed that the ABCG2 Q141K variant was fully processed but retained in the aggresome, a perinuclear structure, where misfolded proteins aggregate. In screening for compounds that could correct its expression, localization, and function, we found that the microtubule-disrupting agent colchicine could induce relocalization of the variant from the aggresome to the cell surface. More strikingly, we found that HDIs could produce a similar effect but also restore protein expression to wild-type levels, yielding a restoration of ABCG2-mediated specific drug efflux activity. Notably, HDIs did not modify aggresome structures but instead rescued newly synthesized protein and prevented aggresome targeting, suggesting that HDIs disturbed trafficking along microtubules by eliciting changes in motor protein expression. Together, these results showed how HDIs are able to restore wild-type functions of the common Q141K polymorphic isoform of ABCG2. More broadly, our findings expand the potential uses of HDIs in the clinic.

Gout, genetics and ABC transporters.

Gout is a chronic arthritic disease associated with high levels of urate in blood. Recent advances in research have permitted the identification of several new and common genetic factors underlying the disease. Among them, a polymorphism in the ABC transporter gene ATP-binding cassette transporter isoform G2 has been highlighted. ATP-binding cassette transporter isoform G2 was found to be involved in renal urate elimination, and the presence of the Q141K polymorphism to induce a 2-fold decrease in urate efflux. The Q141K variant has been shown to have impaired trafficking, leading to its intracellular retention, whereas the wild type protein is expressed on the cell surface. Several agents are being studied for the purpose of improving folding, trafficking and function of various ABC transporters, including ATP-binding cassette transporter isoform G2. If successful, this strategy opens doors to potential new therapies for gout.

Antitumor effect of pharmacologic ascorbate in the B16 murine melanoma model

Because 5-year survival rates for patients with metastatic melanoma remain below 25%, there is continued need for new therapeutic approaches. For some tumors, pharmacologic ascorbate treatment may have a beneficial antitumor effect and may work synergistically with standard chemotherapeutics. To investigate this possibility in melanoma, we examined the effect of pharmacologic ascorbate on B16-F10 cells. Murine models were employed to compare tumor size following treatment with ascorbate, and the chemotherapeutic agents dacarbazine or valproic acid, alone or in combination with ascorbate. Results indicated that nearly all melanoma cell lines were susceptible to ascorbate-mediated cytotoxicity. Compared to saline controls, pharmacologic ascorbate decreased tumor size in both C57BL/6 (P < 0.0001) and NOD-scid tumor bearing mice (P < 0.0001). Pharmacologic ascorbate was superior or equivalent to dacarbazine as an antitumor agent. Synergy was not apparent when ascorbate was combined with either dacarbazine or valproic acid; the latter combination may have additional toxicities. Pharmacologic ascorbate induced DNA damage in melanoma cells, as evidenced by increased phosphorylation of the histone variant, H2A.X. Differences were not evident in tumor samples from C57BL/6 mice treated with pharmacologic ascorbate compared to tumors from saline-treated controls. Together, these results suggest that pharmacologic ascorbate has a cytotoxic effect against melanoma that is largely independent of lymphocytic immune functions and that continued investigation of pharmacologic ascorbate in cancer treatment is warranted.

Histone deacetylase inhibitors induce CXCR4 mRNA but antagonize CXCR4 migration

The stromal cell-derived factor-1α SDF-1α (CXCL12)/CXCR4 axis has been linked to poor prognosis in some cancers. As histone deacetylase inhibitors (HDIs) exert antitumor effects by targeting proteins affecting cell migration, we sought to evaluate the effects of the HDIs apicidin, vorinostat, entinostat (MS-275) and romidepsin on the expression and function of CXCR4 in human cancer cell lines. After treatment with romidepsin, CXCR4 mRNA expression increased 12-fold in UOK121 renal cancer cells, 16-fold in H460 non-small cell cancer cells and 4-fold in SF295 glioma cells; treatment with other HDIs yielded similar effects. CXCR4 induction was not observed in MCF7 breast cancer cells or SW620 colon cancer cells. To evaluate the corresponding functional increase, the effect of CXCR4 ligand, CXCL12, on ERK1/2, STAT3 and c-SRC activation and cell migration was examined in UOK121, SF295 and H460 cells. Alone, the HDIs increased pERK1/2, while reducing pSTAT-3 and pSRC. Following CXCL12 exposure, pERK1/2 induction was maintained, but STAT3 and SRC phosphorylation was impaired. These findings resulted in reduced basal and CXCL12-mediated cell migration. In conclusion, HDIs upregulated CXCR4 mRNA expression but impaired CXCL12-dependent signaling cascades through STAT3 and c-SRC, suggesting a potential role for HDIs in delaying or preventing metastatic processes in solid tumors.

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.

Pancreatic cancer: Targeting KRAS and the vitamin D receptor via microtubules

Pancreatic cancer remains a difficult-to-treat malignancy, yet nab-paclitaxel plus gemcitabine prolongs survival. Closer examination of the mechanism of action of nab-paclitaxel hints at a role for targeting KRAS. We discuss how nab-paclitaxel may be active in pancreatic cancer and how this informs the way forward to better treat patients with pancreatic cancer.

The ABCG2 Multidrug Transporter

ABCG2 is an ATP-binding cassette (ABC) half-transporter localized to the cell membrane and important in normal physiology, in normal tissue protection including in the maternal-fetal barrier and the blood brain barrier, and potentially in multidrug resistance. Dimerization is required for function. Polymorphic variants have been described that impair trafficking to the cell surface, and one, Q141K, has been associated with a higher incidence of gout in individuals who are carriers. Working in concert with ABCB1 (P-glycoprotein), ABCG2 is expressed at high levels in the vascular endothelium of the brain and pumps xenobiotics and chemotherapeutics back into the bloodstream. ABCG2 expression is also found at high levels in some cancer types including pancreatic and liver cancers. Whether its expression in these tumor types contributes to drug resistance via drug efflux and reduction in drug accumulation is a question that remains unanswered. Studies of drug accumulation, such as through imaging solid tumors in patients, are needed to answer this question.