Integrin αV modulates the cellular pharmacology of copper and cisplatin by regulating expression of the influx transporter CTR1.

The αV integrin is expressed in most cancer cells where it regulates a diverse array of cellular functions essential to the initiation, progression and metastasis of solid tumors. However, little is known about how αV integrin modulates cellular sensitivity to chemotherapeutic agents, particularly the platinum drugs. In this study, we found that down-regulation of αV sensitized human M21 cells to cisplatin (cDDP) through up-regulation of the copper influx transporter CTR1. Cells selected for low αV integrin expression (M21L) were more sensitive to cDDP, accompanied by increase in CTR1 mRNA and CTR1 protein levels, more intracellular cDDP accumulation and cDDP DNA adduct formation. Basal copper (Cu) content, Cu uptake, and Cu cytotoxicity were also increased. Transfection of a luciferase reporter construct containing the hCTR1 promoter sequence revealed an increase of the hCTR1 transcription activity in M21L cells. The basis for the increased hCTR1 transcription was related to an increase in the steady-state level of Sp1, a transcription factor known to drive hCTR1 expression. These results indicate that the αV integrin modulates sensitivity of human cells to the cytotoxic effect of cDDP by regulating expression of the Cu transporter CTR1, and introduce the concept that αV expression is linked to Cu homeostasis.

The role of metal binding and phosphorylation domains in the regulation of cisplatin-induced trafficking of ATP7B.

The copper (Cu) exporter ATP7B mediates cellular resistance to cisplatin (cDDP) by increasing drug efflux. ATP7B binds and sequesters cDDP in into secretory vesicles. Upon cDDP exposure ATP7B traffics from the trans-Golgi network (TGN) to the periphery of the cell in a manner that requires the cysteine residues in its metal binding domains (MBD). To elucidate the role of the various domains of ATP7B in its cDDP-induced trafficking we expressed a series of mCherry-tagged variants of ATP7B in HEK293T cells and analyzed their subcellular localization in basal media and after a 1 h exposure to 30 µM cDDP. The wild type ATP7B and a variant in which the cysteines in the CXXC motifs of MBD 1-5 were converted to serines trafficked out of the trans-Golgi (TGN) when exposed to cDDP. Conversion of the cysteines in all 6 of the CXXC motifs to serines, or in only the sixth MBD, rendered ATP7B incapable of trafficking on exposure to cDDP. Truncation of MBD1-5 or MBD1-6 resulted in the loss of TGN localization. Addition of the first 63 amino acids of ATP7B to these variants restored TGN localization to a great extent and enabled the MBD1-5 variant to undergo cDDP-induced trafficking. A variant of ATP7B in which the aspartate 1027 residue in the phosphorylation domain was converted to glutamine localized to the TGN but was incapable of cDDP-induced trafficking. These results demonstrate that the CXXC motif in the sixth MBD and the catalytic activity of ATP7B are required for cDDP-induced trafficking as they are for Cu-induced redistribution of ATP7B; this provides further evidence that cDDP mimics Cu with respect to the molecular mechanisms by they control the subcellular distribution of ATP7B.

Regulation of the Epithelial-Mesenchymal Transition by Claudin-3 and Claudin-4.

The mechanisms that control intracellular adhesion are central to the process of invasion and metastasis. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are major structural molecules of the tight junctions that link epithelial cells. Our prior work has demonstrated that knockdown of the expression of either CLDN3 or CLDN4 produces marked changes in the phenotype of ovarian carcinoma cells including increases in growth rate in vivo, migration, invasion, metastasis, and drug resistance, similar to those produced by the epithelial-to-mesenchymal transition (EMT). We postulated that these changes may result from the ability of CLDN3 or CLDN4 to suppress EMT. In this study we found that knockdown of either CLDN3 or CLDN4 increased cell size and resulted in flattened morphology. While knockdown of CLDN3 or CLDN4 did not alter the expression of vimentin, it significantly down-regulated the level of E-cadherin and up-regulated N-cadherin expression. Conversely, over-expression of CLDN3 or CLDN4 in a cell line that does not express endogenous CLDN3 or CLDN4 decreased N-cadherin expression. Re-expression of E-cadherin in the CLDN3 or CLDN4 knockdown cells reduced migration, invasion and tumor growth in vivo. Loss of either CLDN3 or CLDN4 resulted in activation of the PI3K pathway as evidenced by increased Akt phosphorylation, elevated cellular PIP3 content and PI3K activity as well as up-regulation of the mRNA and protein levels of the transcription factor Twist. Taken together, these findings suggest that CLDN3 and CLDN4 function to sustain an epithelial phenotype and that their loss promotes EMT.

Claudin-3 and claudin-4 regulate sensitivity to cisplatin by controlling expression of the copper and cisplatin influx transporter CTR1.

Claudin-3 (CLDN3) and claudin-4 (CLDN4) are the major structural molecules that form tight junctions (TJs) between epithelial cells. We found that knockdown of the expression of either CLDN3 or CLDN4 produced marked changes in the phenotype of ovarian cancer cells, including an increase in resistance to cisplatin (cDDP). The effect of CLND3 and CLDN4 on cDDP cytotoxicity, cDDP cellular accumulation, and DNA adduct formation was compared in the CLDN3- and CLDN4-expressing parental human ovarian carcinoma 2008 cells and CLDN3 and CLDN4 knockdown sublines (CLDN3KD and CLDN4KD, respectively). Knockdown of CLDN3 or CLDN4 rendered human ovarian carcinoma 2008 cells resistant to cDDP in both in vitro culture and in vivo xenograft model. The net accumulation of platinum (Pt) and the Pt-DNA adduct levels were reduced in CLDN3KD and CLDN4KD cells. The endogenous mRNA levels of copper influx transporter CTR1 were found to be significantly reduced in the knockdown cells, and exogenous expression of CTR1 restored their sensitivity to cDDP. Reexpression of an shRNAi-resistant CLDN3 or CLDN4 up-regulated CTR1 levels, reversed the cDDP resistance, and enhanced TJ formation in the knockdown cells. Baseline copper (Cu) level, Cu uptake, and Cu cytotoxicity were also reduced in CLDN3KD and CLDN4KD cells. Cu-dependent tyrosinase activity was also markedly reduced in both types of CLDN knockdown cells when incubated with the substrate l-DOPA. These results indicate that CLDN3 and CLDN4 affect sensitivity of the ovarian cancer cells to the cytotoxic effect of cDDP by regulating expression of the Cu transporter CTR1.

Tight junction proteins claudin-3 and claudin-4 control tumor growth and metastases.

The extent of tight junction (TJ) formation is one of many factors that regulate motility, invasion, and metastasis. Claudins are required for the formation and maintenance of TJs. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in the majority of ovarian cancers. We report here that CLDN3 and CLDN4 each serve to constrain the growth of human 2008 cancer xenografts and limit metastatic potential. Knockdown of CLDN3 increased in vivo growth rate by 2.3-fold and knockdown of CLDN4 by 3.7-fold in the absence of significant change in in vitro growth rate. Both types of tumors exhibited increase in birth rate as measured by Ki67 staining and decrease in death rate as reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Knockdown of either claudin did not alter expression of other TJ protein but did reduce TJ formation as measured by transepithelial resistance and paracellular flux of dextran, enhance migration and invasion in in vitro assays, and increase lung colonization following intravenous injection. Knockdown of CLDN3 and CLDN4 increased total lung metastatic burden by 1.7-fold and 2.4-fold, respectively. Loss of either CLDN3 or CLDN4 resulted in down-regulation of E-cadherin mRNA and protein, increased inhibitory phosphorylation of glycogen synthase kinase-3ß (GSK-3ß), and activation of ß-catenin pathway signaling as evidenced by increases in nuclear ß-catenin, the dephosphorylated form of the protein, and transcriptional activity of ß-catenin/T-cell factor (TCF). We conclude that both CLDN3 and CLDN4 mediate interactions with other cells in vivo that restrain growth and metastatic potential by sustaining expression of E-cadherin and limiting ß-catenin signaling.

Sec61ß controls sensitivity to platinum-containing chemotherapeutic agents through modulation of the copper-transporting ATPase ATP7A.

The Sec61 protein translocon is a multimeric complex that transports proteins across lipid bilayers. We discovered that the Sec61ß subunit modulates cellular sensitivity to chemotherapeutic agents, particularly the platinum drugs. To investigate the mechanism, expression of Sec61ß was constitutively knocked down in 2008 ovarian cancer cells. Sec61ß knockdown (KD) resulted in 8-, 16.8-, and 9-fold resistance to cisplatin (cDDP), carboplatin, and oxaliplatin, respectively. Sec61ß KD reduced the cellular accumulation of cDDP to 67% of that in parental cells. Baseline copper levels, copper uptake, and copper cytotoxicity were also reduced. Because copper transporters and chaperones regulate platinum drug accumulation and efflux, their expression in 2008 Sec61ß-KD cells was analyzed; ATP7A was found to be 2- to 3-fold overexpressed, whereas there was no change in ATP7B, ATOX1, CTR1, or CTR2 levels. Cells lacking ATP7A did not exhibit increased cDDP resistance upon knockdown of Sec61ß. Sec61ß-KD cells also exhibited altered ATP7A cellular distribution. We conclude that Sec61ß modulates the cytotoxicity of many chemotherapeutic agents, with the largest effect being on the platinum drugs. This modulation occurs through effects of Sec61ß on the expression and distribution of ATP7A, which was shown previously to control platinum drug sequestration and cytotoxicity.

Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency.

BACKGROUND: Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. This study utilized the C-terminal 30 amino acid fragment of C. perfringens enterotoxin (CPE), which binds to claudins 3 and 4, to deliver a toxin in the form of recombinant gelonin (rGel) to the cytoplasm of the human ovarian carcinoma cell line 2008. RESULTS: CPE was fused to rGel at its N-terminal end via a flexible G4S linker. This CPE-G4S-rGel molecule was internalized into vesicles from which location it produced little cytotoxicity. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R9) was introduced between the CPE and the rGel. CPE-R9-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. The addition of a poly-glutamic acid sequence (E9) through a G4S linker to R9-rGel (E9-G4S-R9-rGel) largely neutralized the non-selective cell membrane penetrating activity of the R9 motif. However, introduction of CPE to the E9-G4S-R9-rGel fusion protein (CPE-E9-G4S-R9-rGel) further reduced its cytotoxic effect. Treatment with the endosomolytic reagent chloroquine increased the cytotoxicity of CPE-E9-G4S-R9-rGel. Several types of linkers susceptible to cleavage by furin and endosomal cathepsin B were tested for their ability to enhance R9-rGel release but none of these modifications further enhanced the cytotoxicity of CPE-E9-G4S-R9-rGel. CONCLUSION: We conclude that while a claudin-3 and -4 ligand serves to deliver rGel into 2008 cells the delivered molecules were entrapped in intracellular vesicles. Incorporation of R9 non-specifically increased rGel cytotoxicity and this effect could be masked by inclusion of an E9 sequence. However, the putative protease cleavable sequences tested were inadequate for release of R9-rGel from CPE-E9-G4S-R9-rGel.

Targeting granzyme B to tumor cells using a yoked human chorionic gonadotropin.

PURPOSE: Luteinizing hormone receptor (LHR) is found in abundance on human ovarian, breast, endometrial and prostate carcinomas but at only low levels on non-gonadal tissues. To selectively kill LHR-expressing tumors, granzyme B (GrB) was linked to a protein in which both chains of human chorionic gonadotropin were yoked together (YCG). METHODS: GrB-YCG was expressed and secreted from insect Sf9 cells. Its GrB enzymatic activity and binding affinity for hLHR were then characterized. The differential cytotoxicity of GrB-YCG versus GrB alone was tested in a panel of LHR-expressing tumor cells by SRB assay, and the mechanisms involved in the cell death were investigated by confocal fluorescence microscopy, flow cytometry, and western blot analysis. RESULTS: GrB-YCG was successfully expressed and secreted from Sf9 insect cells and purified from cell culture supernatants. The serine protease activity of GrB-YCG was equivalent to that of human recombinant GrB. An in vitro hormone binding assay revealed that the GrB-YCG molecule also retained the ability to bind to the LHR receptor with an affinity similar to that of native hCG. Upon cell binding, GrB-YCG was rapidly internalized into LHR-expressing human ovarian cancer cells and produced selective and potent tumor cell killing by inducing apoptosis through activation of caspase-3. CONCLUSIONS: These results validate LHR as a therapeutic target and indicate that delivery of the human pro-apoptotic enzyme GrB to tumor cells by yoked hCG has substantial selectivity and therapeutic potential for human tumors that express high levels of LHR such as ovarian carcinomas.

Recombinant CPE fused to tumor necrosis factor targets human ovarian cancer cells expressing the claudin-3 and claudin-4 receptors.

Using gene expression profiling, others and we have recently found that claudin-3 (CLDN3) and claudin-4 (CLDN4) are two of the most highly and consistently up-regulated genes in ovarian carcinomas. Because these tight junction proteins are the naturally occurring receptors for Clostridium perfringens enterotoxin (CPE), in this study, we used the COOH-terminal 30 amino acids of the CPE (CPE(290-319)), a fragment that is known to retain full binding affinity but have no cytolytic effect, to target tumor necrosis factor (TNF) to ovarian cancers. We constructed a pET32-based vector that expressed the fusion protein, designated here as CPE(290-319)-TNF, in which CPE(290-319) was fused to TNF at its NH(2)-terminal end. Western blotting confirmed presence of both CPE(290-319) and TNF in the fusion protein. The TNF component in CPE(290-319)-TNF was 5-fold less potent than free TNF as determined by a standard L-929 TNF bioassay. However, the CPE(290-319)-TNF was >6.7-fold more cytotoxic than free TNF to 2008 human ovarian cancer cells, which express both CLDN3 and CLDN4 receptors. shRNAi-mediated knockdown of either CLDN3 or CLDN4 expression in 2008 markedly attenuated the cytotoxic effects of CPE(290-319)-TNF. The fusion construct was efficiently delivered into target cells and located in both cytosol and vesicular compartments as assessed by immunofluorescent staining. We conclude that CPE(290-319) effectively targeted TNF to ovarian cancer cells and is an attractive targeting moiety for development of CPE-based toxins for therapy of ovarian carcinomas that overexpress CLDN3 and CLDN4.

Acidic hydrolysis of N-Ethoxybenzylimidazoles (NEBIs): potential applications as pH-sensitive linkers for drug delivery.

This paper describes the development of a new class of N-linked imidazoles as potential pH-sensitive, cleavable linkers for use in cancer drug delivery systems. Kinetic analysis of eight derivatives of N-ethoxybenzylimidazoles (NEBIs) showed that their rates of hydrolysis are accelerated in mild aqueous acidic solutions compared to in solutions at normal, physiological pH. Incorporation of electron donating or electron withdrawing substituents on the phenyl ring of the NEBI resulted in the ability to tune the rates of hydrolysis under mild acidic conditions with half-lives ranging from minutes to months. A derivative of NEBI carrying doxorubicin, a widely used anticancer agent, also showed an increased rate of hydrolysis under mild acid compared to that at normal physiological pH. The doxorubicin analogue resulting from hydrolysis from the NEBI exhibited good cytotoxic activity when exposed to human ovarian cancer cells. These results demonstrate a potentially useful, general strategy for conjugating a wide range of drugs to imidazole-containing delivery vessels via NEBI functionalities for controlled release of therapeutics for drug delivery applications.

Novel mechanisms of platinum drug resistance identified in cells selected for resistance to JM118 the active metabolite of satraplatin.

PURPOSE: The goal of this study was to identify molecular determinants of sensitivity and resistance to JM118, the active metabolite of satraplatin, an orally bioavailable cisplatin analog that has activity in prostate cancer. EXPERIMENTAL DESIGN: Human ovarian carcinoma 2008/JM118 cells were derived from parental 2008 cells by repeated exposure to JM118; the revertant 2008/JM118/REV subline was isolated from the 2008/JM118 cells by growth in the absence of drug. Drug sensitivity was determined by clonogenic assay and Pt levels were measured by ICP-MS. RESULTS: Eight sequential rounds of selection yielded the 2008/JM118 subline that was 4.9-fold resistant to JM118 and cross-resistant at varying levels to satraplatin, cisplatin, carboplatin, and oxaliplatin. Cross-resistance to the other Pt drugs was lost as resistance to JM118 waned. The same parental 2008 cells selected for resistance to cisplatin were partially cross-resistant to JM118. The 2008/JM118 cells accumulated significantly more Pt than the 2008 cells when exposed to low concentrations of either JM118 or cisplatin indicating a detoxification process that involves intracellular sequestration. In contrast, 2008 cells selected for cisplatin resistance accumulated less cisplatin and less JM118 reflecting a mechanism involving reduced accumulation. The 2008 and 2008/JM118 cells did not differ in their uptake or efflux of 64Cu, expression of Cu efflux transporters ATP7A or ATP7B or their glutathione content. The 2008/JM118 cells exhibited 3.0-7.7-fold hypersensitivity to docetaxel, paclitaxel and doxorubicin. Expression profiling identified 4 genes that were significantly up-regulated and 19 that were down-regulated in the 2008/JM118 cells at a false discovery rate of 1 gene. CONCLUSIONS: While the cellular defense mechanisms that protect cells against JM118 also mediate resistance to the other Pt drugs, these mechanisms are quite different from those commonly found in cells selected for resistance to cisplatin. JM118-resistant cells accumulate more rather than less Pt and rely on an intracellular detoxification mechanism different from that involved in cisplatin resistance. This is consistent with clinical evidence suggesting that satraplatin has activity in diseases in which cisplatin does not. In this model, JM118 resistance is associated with substantial collateral hypersensitivity to docetaxel, paclitaxel, and doxorubicin.

Contribution of the major copper influx transporter CTR1 to the cellular accumulation of cisplatin, carboplatin, and oxaliplatin.

The goal of this study was to determine the ability of the major copper influx transporter CTR1 to mediate the cellular accumulation of cisplatin (DDP), carboplatin (CBDCA), and oxaliplatin (L-OHP). Wild-type murine embryonic fibroblasts (CTR1+/+) and a subline in which both alleles of CTR1 were deleted (CTR1-/-) were tested for their ability to accumulate platinum when exposed to increasing concentrations of DDP, CBDCA, or L-OHP for 1 h. They were also tested for their sensitivity to the growth-inhibitory effect of each drug. Platinum content was measured by ion-coupled plasmon mass spectroscopy. The experimental model was validated by measuring copper accumulation and cytotoxicity. CTR1-/- cells accumulated only 5.7% as much copper as CTR1+/+ cells during a 1-h exposure to 2 microM copper. When exposed to DDP, CBDCA, or L-OHP at 2 microM, accumulation in the CTR1-/- cells was only 35 to 36% of that in the CTR1+/+ cells. When tested at a 5-fold higher concentration, this deficit remained for DDP and CBDCA, but accumulation of L-OHP was no longer CTR1-dependent. There was an association between the effect of loss of CTR1 function on uptake of the platinum drugs and their cytotoxicity. The CTR1-/- cells were 3.2-fold resistant to DDP, 2.0-fold resistant to CBDCA, but only 1.7-fold resistant to L-OHP. Thus, whereas CTR1 controls the cellular accumulation of all three drugs at low concentrations, accumulation of L-OHP is not dependent on CTR1 at higher concentrations. We conclude that L-OHP is a substrate for some other cellular entry mechanism, a feature consistent with its different clinical spectrum of activity.

Identification of genes whose expression is associated with cisplatin resistance in human ovarian carcinoma cells.

The goal of this study was to identify genes consistently differentially expressed in multiple pairs of isogenic cisplatin (DDP)-sensitive and resistant human ovarian carcinoma cell lines using microarray-based expression profiling. Expression profiling was carried out on six pairs of ovarian carcinoma cells lines growing under identical conditions; each cell expression profile was independently replicated six times. No genes were differentially expressed in all six pairs of cells or even in even in any five of the six pairs. Eighteen genes and 1 EST were upregulated, and four genes and 1 EST were downregulated, in at least four cell pairs. Of these, only metallothionein 2A has previously been implicated in DDP resistance. Among the genes identified on the basis of six replicates, an average of 24.8% would have been missed if only five replicates had been performed, and 38.3% would have been missed with only four replicates. The genes did not identify a dominant biochemical pathway or ontology category as being linked to DDP resistance; however, hierarchical clustering provided evidence for two classes DDP-resistant phenotypes within which there are additional cell pair-specific alterations. Many of the genes identified in this study play important roles in cell surface interactions and trafficking pathways not previously linked to DDP resistance. The genes discovered by this extensively replicated analysis are candidates for prediction of DDP responsiveness in ovarian cancer patients.

cDNA microarray-based identification of genes and pathways associated with oxaliplatin resistance.

In order to identify genes whose expression is associated with resistance to the chemotherapeutic agent oxaliplatin, transcripts differentially expressed between an oxaliplatin sensitive and a stably resistant subline were compared in six independent replicates using Stanford cDNA microarrays for five cell lines. "Significance analysis of microarrays" (SAM) was used to identify genes whose expression was statistically significantly different in the sensitive versus resistant members of each cell line pair. The biochemical pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were searched to identify those pathways in which the number of SAM-identified genes exceeded the number expected. This identified four pathways in which upregulated genes were significantly associated with resistance in two of the cell line pairs, and two pathways in which the association was found in three cell line pairs. The search also identified 12 pathways in which downregulated genes were associated with resistance in two cell line pairs and one pathway in which the association reached statistical significance in three cell line pairs. Pathways identified included the ribosome pathway, the Huntington's disease pathway that includes caspase 8, and the ATP synthesis pathways. Determination of the chromosomal location of each SAM-identified gene revealed several locales within which genes lay in close proximity, including three genes (APACD, IF-2, and REV1L) located on chromosome 2 that lie immediately adjacent to each other and were significantly upregulated in three of five cell line pairs. Biochemical pathway and chromosomal mapping of genes identified by SAM as differentially expressed in related cell line pairs points to mechanisms and chromosomal sites not previously suspected of association with the oxaliplatin-resistant phenotype.

Identification of transdominant-negative genetic suppressor elements derived from hMSH2 that mediate resistance to 6-thioguanine.

Using random screening for genetic suppressor elements, we sought to identify portions of hMSH2 important to the ability of the mismatch repair system to recognize and process DNA adducts that mimic mismatches. All recovered candidate genetic suppressor elements were derived from the region containing amino acids 782 to 844. Expression of a peptide corresponding to this region partially disabled mismatch repair as evidenced by 1.5- to 3.3-fold resistance to 6-thioguanine, cisplatin, and N-methyl-N'-nitrosoguanidine, an increase in the rate of generation of drug resistant variants, and the appearance of microsatellite instability. Even low-level expression of this protein was sufficient to partially impair mismatch repair. The results suggest that this region is important to the ability of the mismatch repair system to mediate drug sensitivity and to maintain genomic stability.

Using mRNA expression profiling to determine anticancer drug efficacy.

Pharmacogenomics is a fast-growing field of investigations that aims to further elucidate the inherited nature of interindividual differences in drug disposition and effects, with the ultimate goal of providing a stronger scientific basis for selecting the optimal drug therapy. Providing the right drug for the right patient is an important problem in the treatment of cancer. This is mainly due to the lack of information about the sensitivity of the tumor for a specific treatment modality, such as either chemotherapy or radiation treatment. This presentation highlights two approaches to identify responsiveness to treatment. Both approaches are based on the identification of expression profiles. The first approach concentrates on drug resistance and the second on the signaling pathways leading up to the death of the cell. Both approaches provide expression profiles; however, the more dynamic expression profiling as used to determine the signaling in damage cells promises to be a better determinant for the pharmacogenomic changes in expression profiles and, consequently, a potential better determinant for drug efficacy.