Victor Ling

Multidrug resistance: molecular mechanisms and clinical relevance

Abstractu2003Multidrug resistance (MDR) describes the phenomenon of simultaneous resistance to unrelated drugs. It has been a decade since the P-glycoprotein (Pgp) gene, which is associated with a form of MDR caused by reduced drug accumulation, was cloned. Thus, this would seem to be an appropriate time to evaluate our understanding of this form of MDR. The two MDR genes identified in humans to date (the MDR-associated protein [MRP] and Pgp genes) are structurally similar and both are members of the ATP-binding cassette (ABC) transporter family. Although the physiological role of MRP is not yet understood, one Pgp gene (mdr1) plays an important role in the blood-tissue barrier and the other (mdr2/3) is involved in phospholipid transport in the liver. A variety of compounds (chemosensitizing agents) can interfere with Pgp and MRP function; such agents may improve the efficacy of conventional therapy when used in combination with such regimens. Determining the roles cellular MDR mechanisms play in patients’ response to chemotherapy is a major challenge. Using Pgp and MRP as molecular markers to detect MDR tumor cells is technically demanding, and solid tumors in particular contain heterogeneous cell populations. Since MDR requires Pgp or MRP gene expression, clinically relevant gene expression thresholds need to be established; sequential samples from individual patients are valuable for correlating MDR gene expression with the clinical course of disease. Studies in leukemias, myelomas, and some childhood cancers show that Pgp expression correlates with poor response to chemotherapy. However, in some cases, inclusion of a reversing or chemosensitizing agent such as verapamil or cyclosporin A has improved clinical efficacy. Such agents may inactivate Pgp in tumor cells or affect Pgp function in normal cells, resulting in altered pharmacokinetics. It would be interesting to determine whether patients who fail treatment in the presence of chemosensitizing agents acquire other MDR mechanisms. The ABC transporter superfamily in prokaryotes and eukaryotes is involved in the transport of substrates ranging from ions to large proteins. Of the 15 or more ABC transporter genes characterized in human cells, two (Pgp and MRP) cause MDR. Therefore, it would be relevant to determine the number of such genes present in the human genome; however, extrapolating from the number of ABC transporter genes in bacteria, the human gene probably contains a minimum of 200 ABC transporter superfamily members. Thus, tumor cells can potentially use many ABC transporters to mount resistance to known and future therapeutic agents. The challenge will be to determine which ABC transporters are clinically relevant. Despite the potential of tumor cells to protect themselves, a variety of malignancies can be successfully treated with chemotherapy. This may provide unique insights.

Patient-Derived First Generation Xenografts of Non–Small Cell Lung Cancers: Promising Tools for Predicting Drug Responses for Personalized Chemotherapy

Purpose: Current chemotherapeutic regimens have only modest benefit for non–small cell lung cancer (NSCLC) patients. Cumulative toxicities/drug resistance limit chemotherapy given after the first-line regimen. For personalized chemotherapy, clinically relevant NSCLC models are needed for quickly predicting the most effective regimens for therapy with curative intent. In this study, first generation subrenal capsule xenografts of primary NSCLCs were examined for (a) determining responses to conventional chemotherapeutic regimens and (b) selecting regimens most effective for individual patients. Experimental Design: Pieces (1×3×3 mm3) of 32 nontreated, completely resected patients NSCLCs were grafted under renal capsules of nonobese diabetic/severe combined immunodeficient mice and treated with (A) cisplatin+vinorelbine, (B) cisplatin+docetaxel, (C) cisplatin+gemcitabine, and positive responses (treated tumor area ≤50% of control, P < 0.05) were determined. Clinical outcomes of treated patients were acquired. Results: Xenografts from all NSCLCs were established (engraftment rate, 90%) with the retention of major biological characteristics of the original cancers. The entire process of drug assessment took 8 weeks. Response rates to regimens A, B, and C were 28% (9 of 32), 42% (8 of 19), and 44% (7 of 16), respectively. Certain cancers that were resistant to a particular regimen were sensitive to others. The majority of responsive tumors contained foci of nonresponding cancer cells, indicative of tumor heterogeneity and potential drug resistance. Xenografts from six of seven patients who developed recurrence/metastasis were nonresponsive. Conclusions: Models based on first generation NSCLC subrenal capsule xenografts have been developed, which are suitable for quick assessment (6-8 weeks) of the chemosensitivity of patients cancers and selection of the most effective regimens. They hold promise for application in personalized chemotherapy of NSCLC patients. Clin Cancer Res; 16(5); 1442–51

The xc- cystine/glutamate antiporter: a mediator of pancreatic cancer growth with a role in drug resistance.

The xc− cystine transporter enhances biosynthesis of glutathione, a tripeptide thiol important in drug resistance and cellular defense against oxidative stress, by enabling cellular uptake of cystine, a rate-limiting precursor. Because it is known to regulate glutathione levels and growth of various cancer cell types, and is expressed in the pancreas, we postulate that it is involved in growth and drug resistance of pancreatic cancer. To examine this, we characterised expression of the xc− transporter in pancreatic cancer cell lines, MIA PaCa-2, PANC-1 and BxPC-3, as subjected to cystine-depletion and oxidative stress. The results indicate that these cell lines depend on xc−-mediated cystine uptake for growth, as well as survival in oxidative stress conditions, and can modulate xc− expression to accommodate growth needs. Immunohistochemical analysis showed that the transporter was differentially expressed in normal pancreatic tissues and overexpressed in pancreatic cancer tissues from two patients. Furthermore, gemcitabine resistance of cells was associated with elevated xc− expression and specific xc− inhibition by monosodium glutamate led to growth arrest. The results suggest that the xc− transporter by enhancing glutathione biosynthesis plays a major role in pancreatic cancer growth, therapy resistance and represents a potential therapeutic target for the disease.

MDR1 and BCRP1 expression in leukemic progenitors correlates with chemotherapy response in acute myeloid leukemia

OBJECTIVEnOverexpression of members of the adenosine triphosphate binding cassette (ABC) transporter superfamily has been implicated in multidrug resistance in cancer, but results in acute myeloid leukemia (AML) have been inconsistent. We investigated the expression and activity of ABC transporters in patient total blasts and subpopulations along the leukemic stem cell hierarchy.nnnMATERIALS AND METHODSnUsing quantitative reverse transcriptase polymerase chain reaction, we measured expression of the ABC transporter superfamily in the blast cells from AML patients prior to chemotherapy. In addition, we measured ex vivo daunorubicin resistance of subpopulations with or without ABC inhibitors.nnnRESULTSnIn the total blasts, no consistent difference was observed in 18 patients achieving complete remission (CR) and 13 patients who were refractory to induction chemotherapy (NR). However, among the subpopulation of CD34(+)CD38(-) AML cells (candidate leukemic stem cells), elevated expression of MDR1 and/or BCRP1, two ABC transporters associated with drug resistance, was found in 8 of 10 NR patients as compared to 0 of 7 CR patients. No such association was observed in the more differentiated CD34(+)CD38(+) or CD34(-) subpopulations. There was no significant difference in MRP1 expression between CR and NR patient samples in any of the subpopulations examined. The increased expression of MDR1 and BCRP1 in leukemic cells correlated with increased cellular daunorubicin resistance, which could be reversed by the ABC transporter inhibitors verapamil and PSC-833.nnnCONCLUSIONnExpression of MDR1 and BCRP1 in leukemic stem cells correlates with chemotherapy response both at the cellular level and in AML patients.

ASAP1, a Gene at 8q24, Is Associated with Prostate Cancer Metastasis

Metastatic prostate cancer is a terminal disease, and the development of reliable prognostic tools and more effective therapy is critically important for improved disease survival and management. This study was aimed at identifying genes that are differentially expressed in metastatic and nonmetastatic prostate cancer cells and, as such, could be critical in the development of metastasis. Long-SAGE analysis was used to compare a transplantable human metastatic prostate cancer subline, PCa1-met, with a nonmetastatic counterpart, PCa2. Both sublines were developed from a patients prostate cancer specimen via subrenal capsule grafting and subsequent orthotopic implantation into SCID mice. Among various differentially expressed genes identified, ASAP1, an 8q24 gene encoding an ADP-ribosylation factor GTPase-activating protein not previously associated with prostate cancer, was up-regulated in the metastatic subline as confirmed by quantitative real-time PCR. Immunohistochemistry of xenograft sections showed that cytoplasmic ASAP1 protein staining was absent or weak in benign tissue, significantly stronger in nonmetastatic PCa2 tissue, and strongest in PCa1-met tissue. In clinical specimens, ASAP1 protein staining was elevated in 80% of primary prostate cancers and substantially higher in metastatic lesions compared with benign prostate tissue. Moreover, additional ASAP1 gene copies were detected in 58% of the primary prostate cancer specimens. Small interfering RNA-induced reduction of ASAP1 protein expression markedly suppressed in vitro PC-3 cell migration (approximately 50%) and Matrigel invasion (approximately 67%). This study suggests that the ASAP1 gene plays a role in prostate cancer metastasis and may represent a therapeutic target and/or biomarker for metastatic disease.

Compensatory role of P-glycoproteins in knockout mice lacking the bile salt export pump†

Bile salt export pump (BSEP; ATP‐binding cassette, subfamily B, member 11) mutations in humans result in progressive familial intrahepatic cholestasis type 2, a fatal liver disease with greatly reduced bile flow. However in mice, Bsep knockout leads only to mild cholestasis with substantial bile flow and up‐regulated P‐glycoprotein genes (multidrug resistance protein 1a [Mdr1a] and Mdr1b). To determine whether P‐glycoprotein is responsible for the relatively mild phenotype observed in Bsep knockout mice, we have crossed mouse strains knocked out for Bsep and the two P‐glycoprotein genes and generated a triple knockout mouse. We found that a knockout of the three genes leads to a significantly more severe phenotype with impaired bile formation, jaundice, flaccid gallbladder, and increased mortality. The triple knockout mouse is the most severe genetic model of intrahepatic cholestasis yet developed. Conclusion: P‐glycoprotein functions as a critical compensatory mechanism, which reduces the severity of cholestasis in Bsep knockout mice. (HEPATOLOGY 2009.)

Same-single-cell analysis for the study of drug efflux modulation of multidrug resistant cells using a microfluidic chip

Since multidrug resistance (MDR) is a major cause of failure in cancer chemotherapy, we report a microfluidic approach combined with the same-single-cell analysis to investigate the modulation of MDR, manifested as the inhibition of drug efflux. A microfluidic chip that was capable of selecting and retaining a single multidrug-resistant cancer cell was used to investigate drug efflux inhibition in leukemia cell lines. Three advantages of the microfluidic-based same-single-cell analysis (dubbed as SASCA) method have been revealed. First, it readily detects the modulation of drug efflux of anticancer compounds (e.g., daunorubicin) by MDR modulators (e.g., verapamil) among cellular variations. Second, SASCA is able to compare the different cellular abilities in response to drug efflux modulation based on the drug transport kinetics of single cells. Third, SASCA requires only a small number of cells, which may be beneficial for investigating drug resistance in minor cell subpopulations (e.g., cancer stem cells).

INCREASED P-GLYCOPROTEIN MESSENGER RNA STABILITY IN RAT LIVER TUMORS IN VIVO

P‐glycoproteins (Pgp) are comprised of a small family of plasma membrane proteins whose abundance in cultured cells is often associated with the multidrug resistance phenotype. Overexpression of Pgp has been observed in many types of human cancers, but the molecular basis for this overexpression has not been established. We have used primary monolayer cultures of adult rat hepatocytes and a stepwise model of rat liver carcinogenesis to study the regulation of Pgp gene expression. We observed a marked overexpression of Pgp, specifically the class II Pgp, in both systems. In addition, we observed that a number of unrelated genes including α‐tubulin, β‐actin, γ‐actin, cytokeratin 8, cytokeratin 18, and c‐myc are overexpressed in cultured hepatocytes, and they are also overexpressed during liver carcinogenesis and in transplantable tumors. Nuclear run‐on assays showed no increase in the transcriptional activity of Pgp genes in transplantable liver tumors compared to normal liver. Studies of in vivo mRNA stability, however, revealed that all three Pgp mRNAs were relatively stable in transplantable liver tumors (t1/2 > 12 h), in contrast to what was found in normal liver (t1/2 < 2 h). In addition, mRNA for several other genes, including α‐tubulin, c‐myc, and cyclin D1, all appear to be stabilized in the tumors. These findings suggest that the overexpression of Pgp genes in rat liver tumors may be the result of a mechanism involving stabilization of a diverse group of mRNAs. J. Cell. Physiol. 177:1–12, 1998.

Preface: the concept and consequences of multidrug resistance

The problem of multidrug resistance (MDR) in human cancers led to the discovery 30xa0years ago of a single protein P-glycoprotein (P-gp), capable of mediating resistance to multiple structurally diverse drugs. P-gp became the archetypal eukaryotic ABC transporter gene, and studies of P-gp and related ABC transporters in both eukaryotes and bacteria have led to a basic mechanistic understanding of the molecular basis of MDR. Particular milestones along the way have been the identification of the homology between P-gp and bacterial transport proteins, the purification and functional reconstitution of P-gp into synthetic lipid systems, and the development of targeted therapies that attempt to overcome MDR by inhibiting P-gp. This preface places into this context some of the less well-explored themes developed in the MDR field, particularly various alternative models of P-gp action, evidence for parallel physiological roles for P-gp, and the unusual relationship between the substrate recognition capabilities of ABC transporters and their evolutionary history.

Genetic changes in the evolution of multidrug resistance for cultured human ovarian cancer cells

The multidrug resistant (MDR) phenotype is often attributed to the activity of ATP‐binding cassette (ABC) transporters such as P‐glycoprotein (ABCB1). Previous work has suggested that modulation of MDR may not necessarily be a single gene trait. To identify factors that contribute to the emergence of MDR, we undertook integrative genomics analysis of the ovarian carcinoma cell line SKOV3 and a series of MDR derivatives of this line (SKVCRs). As resistance increased, comparative analysis of gene expression showed conspicuous activation of a network of genes in addition to ABCB1. Functional annotation and pathway analysis revealed that many of these genes were associated with the extracellular matrix and had previously been implicated in tumor invasion and cell proliferation. Further investigation by whole genome tiling‐path array CGH suggested that changes in gene dosage were key to the activation of several of these overexpressed genes. Remarkably, alignment of whole genome profiles for SKVCR lines revealed the emergence and decline of specific segmental DNA alterations. The most prominent alteration was a novel amplicon residing at 16p13 that encompassed the ABC transporter genes ABCC1 and ABCC6. Loss of this amplicon in highly resistant SKVCR lines coincided with the emergence of a different amplicon at 7q21.12, which harbors ABCB1. Integrative analysis suggests that multiple genes are activated during escalation of drug resistance, including a succession of ABC transporter genes and genes that may act synergistically with ABCB1. These results suggest that evolution of the MDR phenotype is a dynamic, multi‐genic process in the genomes of cancer cells.