Colorectal cancer is the third most common form of cancer and the second leading cause of cancer-related death in the Western world. Colon cancer causes 655,000 deaths worldwide per year [1]. Therapy is usually through surgery, followed in many cases by chemotherapy, which is used to slow tumor growth, to shrink tumor size and to reduce the likelihood of metastasis development.

Chemotherapy effectiveness in cancer cells is compromised by the achievement of drug resistance. Therefore, gaining insight into the mechanisms underlying drug resistance is basic to develop more effective therapeutic approaches. Morales et al. [2] hypothesized that the genetic features related with the progression pathway in colorectal cancer may condition its chemoresistance capability. In fact, it has been described that the tumor's ability to survive, grow and metastasize is conditioned by its genetic and phenotypic heterogeneity [3].

Methotrexate (MTX) is an antimetabolite and antifolate drug used in treatment of cancer and autoimmune diseases. MTX competitively and reversibly inhibits dihydrofolate reductase (DHFR), an enzyme that participates in folate metabolism, and essential for DNA synthesis and cell growth [4]. MTX is used for the treatment of lymphoblastic leukemia, lymphoma, osteosarcoma, breast cancer, and head and neck cancer [5]. Treatments combining MTX and other drugs are used in colorectal cancer [6–8]. However, MTX resistance can be easily acquired through different ways, although amplification of the target gene (dhfr) has been shown to be the most important mechanism of resistance in cultured cells [9–11]. Indeed, amplification of 5q12-14 regions, where dhfr is located, has been described in MTX-resistant HT29 cells [2].

In the present study, we wanted to identify genes implicated in MTX resistance in HT29 colon cancer cells and to explore their relative contribution to this phenotype. We analyzed the differential gene expression between MTX-resistant and MTX-sensitive HT29 cells using oligonucleotide microarrays containing the full human genome. Changes in the DNA content between both cell lines were also determined. We showed a role for specific differentially expressed genes in MTX resistance. Using siRNAs against caveolin 1, enolase 2 and PKCα or plasmid overexpression for E-cadherin, a clear chemosensitization toward MTX was observed.

In the present study, genes differentially expressed in HT29 colon cancer cells resistant to MTX were identified and their relative contribution to this phenotype evaluated. We observed a cluster of genes flanking the dhfr locus in chromosome 5 that were overexpressed in MTX-resistant HT29 cells. Two of the genes included in this cluster, MSH3 and XRCC4, are known to be involved in DNA repair [17–19]; other two, RASGRF2 and SSBP2, have been related to signaling pathways [20–22]; and EDIL3 has been suggested to prevent apoptosis and to promote cell proliferation [23, 24]. Despite the confirmation of the co-amplification of all these genes with dhfr in the resistant cells, we did not observe a clear sensitization toward MTX when reducing their respective mRNA levels by means of iRNA technology. Our observations indicate that the increase in copy-number and the resulting upregulation of the studied genes in 5q14 may be a consequence of dhfr amplification more than an adaptation of the cells to MTX resistance. Indeed, many mammalian species (mouse, rat, bull, cock, dog and chimpanzee) show this set of genes in the same order around dhfr as in human chromosome 5 (using the MapViewer at NCBI), indicating a conserved pattern of gene organization. In keeping with this, its overexpression in the resistant cells could have been useful to improve some cellular processes that might facilitate survival. However, as shown in this work, the increase in copy number of this set of genes does not favor MTX resistance. Thus, we decided to search for other differentially expressed genes (CAV1, E-cadherin, ENO2 and PKCα) that had been previously related with resistance or with colon cancer and to evaluate their relative contribution in our cell system.

Enolase 2 (ENO2) is induced by hypoxia, an intrinsic condition of tumors. Moreover, ENO2 is a glycolysis-related gene that has been described to play an important role in tumorogenesis of colorectal cancers [25]. Indeed, ENO2 is upregulated in a variety of cancers [26–28] and alpha-enolase is significantly upregulated in a metastasic colon cancer cell line, suggesting a possible association with the metastasic process in vitro and in vivo [29]. Indeed, we observed a notable contribution of ENO2 to MTX resistance when treating the sensitive cells with siENO2.

Both the α-isozyme of PKC and caveolin 1 has been described to be associated with multidrug resistance [30, 31], and thus represent good targets to be analyzed. PKCα phosphorylates different proteins, which triggers a wide variety of cellular responses including proliferation, differentiation, membrane transport, gene expression and tumor promotion [32, 33]. Chemical inhibitors of PKC activity have been proposed as resistance modulators in MTX chemotherapy [34]. Furthermore, decreasing PKCα mRNA levels attenuates the MDR phenotype in tumor cells [35] and increases the sensitivity to anticancer drugs, both in vitro [36–38] and in vivo [39]. These observations are in accordance with our result showing that the decrease of PKCα mRNA levels by means of iRNA technology causes a sensitization of the cells toward MTX. Caveolin 1 (CAV1), the principal component of caveolae, has been associated with progression of colon and breast carcinomas [40, 41] and with enhanced invasiveness in lung adenocarcinoma cells [42]. Although suggested as tumor suppressor gene, and downregulated in some oncogene-transformed and tumor-derived cells [31], overexpression of CAV1 has been found in prostate and esophageal cancer [43–45]. Moreover, re-expression of CAV1 at latter stages of tumor development has been described in human and mouse prostate adenocarcinomas [41], a scenario that could resemble chemotherapy resistance. Indeed, Bender et al. [46] found significantly higher levels of CAV1 in MTX resistant HT29 clones. We have confirmed the implication of CAV1 in MTX resistance in our HT29 cell line.

Nevertheless, as Benimetskaya and collaborators observed with PKCα [47], downregulation of a gene alone may be insufficient to completely chemosensitize the cells. Therefore, we considered a combination therapy in order to improve MTX sensitivity. As shown in figure 5, the combination of siRNAs against CAV1, ENO2 and PKCα sensitizes the cells toward MTX, and the effect is improved by the additional downregulation of DHFR. The effects of the triple or the quadruple combinations, however, are not the sum of the effects caused by each single siRNA. This probably reflects the difficulty of transfecting more than one siRNA at 100 nM each. Indeed, the mRNA levels for the four genes after the combination treatment were not as reduced as with the single treatments. In the case of all treatments performed in the resistant cells, probably the overexpression by amplification of the dhfr locus was powerful enough to mask the effects of the siRNAs used.

Not only the overexpression of some genes may cause the resistance phenotype. One of the most underexpressed genes that we confirmed to be clearly lost in our HT29 MTX-resistant cells is E-cadherin. In fact, loss of E-cadherin, frequently observed in epithelial tumors, has been associated with tumor progression [48, 49] and is considered a crucial event that favors metastasis and invasiveness [50, 51]. In addition, the mRNA levels of E-cadherin in adenocarcinoma are 2-fold lower than in normal colon cells [52]. Thus, there is a functional correlation between E-cadherin levels and malignancy. It has been described an event of loss of heterozygosity at the 16.1q chromosome band in most human prostate cancers, where E-cadherin is located [53], which is associated with tumor grade, advanced clinical stage and poor survival [54]. Our experiments show a decrease of 3-fold in E-cadherin levels in resistant cells and also that a mild overexpression of E-cadherin causes a higher sensitivity toward MTX. One has to be cautious, however, about the expression levels of E-cadherin since an increase of more than 3-fold in any of both cell lines caused a reduction in cell viability. This is in accordance with the experiments of Derksen et al. [55] that suggested that loss of E-cadherin could play a causal role in the acquisition of anoikis resistance, as parental mammary cells lacking E-cadherin survived while re-expression of the gene caused apoptosis [55]. Previous works show that loss of E-cadherin in either skin or mammary epithelium does not induce tumor formation [55]. Thus, an overall view of the events occurring in our HT29 cells resistant to methotrexate is needed.

It has been shown that activated PKCα translocates from the nucleus to the membrane [56], where it associates with caveolae [57, 58], and regulates the function and formation of such biological structures. PKCα has been described to directly interact with CAV1. The union is performed between the caveolin 1 scaffolding domain peptide and PKCα caveolin 1 binding motif [59]. Further, activation of PKCα by phorbol esters dislocates the enzyme from caveolae. All these observations indicate that PKCα interacts functionally with this membrane structures. Moreover, PKCα has been proposed to be involved in the rearrangement of the cytoskeleton. Masur et al. showed that a high level of PKCα expression plus a low E-cadherin level predicts an elevated migratory activity of colon carcinoma cells, which could be derived more easily to metastasis [56]. Lahn et al. speculate that PKCα overexpression may represent an important cellular event leading to enhanced tumor progression, as they concluded that MCF-7 breast cancer cells transfected with PKCα had reduced expression of E-cadherin and β-catenin, resulting in a loss of cell-cell adhesion and thus in a more aggressive tumor phenotype [38].

Specific protein-protein interactions between CAV1 and other proteins have been proposed to regulate cell signaling [57, 60]. Indeed, CAV1 is known to control cell proliferation and viability by inhibiting expression of survivin, a member of the IAP (inhibitor of apoptosis) family via a transcriptional mechanism involving the β-catenin-Tcf/Lef-1 pathway [16]. One of the possible locations of β-catenin is within a complex with E-cadherin in the adherence junctions, specialized cell-cell adhesion sites that link the cadherin molecules to the actin microfilaments [61]. E-cadherin promotes co-localization and co-imunoprecipitation of CAV1 with β-catenin, as well as inhibition of β-catenin-Tcf/Lef-1 dependent transcription of a wide variety of genes regulated by this pathway, among which survivin is found. However, the ability of CAV1 to regulate survivin expression and cell proliferation is severely impaired in metastasic cancer cells lacking E-cadherin [16]. If E-cadherin is lost, β-catenin is not retained in the plasma membrane and can be then translocated into the nucleus [62], thus activating Tcf/Lef-1 transcription factors-mediated expression of genes implicated in cell proliferation and tumor progression [50]. E-cadherin has been shown to be an important permissive element in defining the functions of CAV1, since several characteristics potentially relevant to CAV1 function as a tumor suppressor are compromised in E-cadherin-deficient HT29 cells [16]. A diagram showing all these relations is presented in Figure 8.

Our results show that HT29 cells can be well sensitized toward MTX by simultaneous treatment with siCAV1 and pBATEM2-CDH. Importantly, we can revert the resistant scenario by reducing the levels of caveolin 1 and by overexpressing E-cadherin simultaneously in the resistant cells, demonstrating the roles that play both genes in MTX resistance.

We demonstrate that, aside from dhfr, the contribution of the 5q14 co-amplified genes to MTX resistance is small in HT29 colon cancer cells. On the other hand, we have identified genes deregulated in MTX resistant cells and have demonstrated a role for caveolin 1, E-cadherin, enolase 2 and PKCα in MTX resistance. Very importantly, the concomitant knocking down of CAV1 with overexpression of E-cadherin in HT29 resistant cells markedly reduced cell viability.