Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells

Elisabet Selga,Cristina Morales,Véronique Noé,Carlos J Ciudad,Miguel A Peinado

doi:10.1186/1755-8794-1-35

Abstract

BackgroundMethotrexate is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment with methotrexate is limited by the development of drug resistance, which may be acquired through different ways. To get insights into the mechanisms associated with drug resistance and sensitization we performed a functional analysis of genes deregulated in methotrexate resistant cells, either due to its co-amplification with the dhfr gene or as a result of a transcriptome screening using microarrays.MethodsGene expression levels were compared between triplicate samples from either HT29 sensitive cells and resistant to 10-5 M MTX by hybridization to the GeneChip® HG U133 PLUS 2.0 from Affymetrix. After normalization, a list of 3-fold differentially expressed genes with a p-value < 0.05 including multiple testing correction (Benjamini and Hochberg false discovery rate) was generated. RT-Real-time PCR was used to validate the expression levels of selected genes and copy-number was determined by qPCR. Functional validations were performed either by siRNAs or by transfection of an expression plasmid.ResultsGenes adjacent to the dhfr locus and included in the 5q14 amplicon were overexpressed in HT29 MTX-resistant cells. Treatment with siRNAs against those genes caused a slight reduction in cell viability in both HT29 sensitive and resistant cells. On the other hand, microarray analysis of HT29 and HT29 MTX resistant cells unveiled overexpression of caveolin 1, enolase 2 and PKCα genes in resistant cells without concomitant copy number gain. siRNAs against these three genes effectively reduced cell viability and caused a decreased MTX resistance capacity. Moreover, overexpression of E-cadherin, which was found underexpressed in MTX-resistant cells, also sensitized the cells toward the chemotherapeutic agent. Combined treatments targeting siRNA inhibition of caveolin 1 and overexpression of E-cadherin markedly reduced cell viability in both sensitive and MTX-resistant HT29 cells.ConclusionWe provide functional evidences indicating that caveolin 1 and E-cadherin, deregulated in MTX resistant cells, may play a critical role in cell survival and may constitute potential targets for coadjuvant therapy.

Highlights

Methotrexate is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders
Identification of genes deregulated in association with MTX resistance The expression profile of the 47,000 transcripts and variants included in the HG U133 PLUS 2.0 microarray from Affymetrix was compared between HT29 sensitive cells and resistant to 10-5 M MTX
The set of upregulated genes in this location included dhfr, zfyve16, msh3, rasgrf2, ssbp2, xrcc4, hapln1 and edil3 (Figure 2), which were selected for further studies

Summary

Introduction

Methotrexate is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment with methotrexate is limited by the development of drug resistance, which may be acquired through different ways. To get insights into the mechanisms associated with drug resistance and sensitization we performed a functional analysis of genes deregulated in methotrexate resistant cells, either due to its co-amplification with the dhfr gene or as a result of a transcriptome screening using microarrays. 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]. Chemotherapy effectiveness in cancer cells is compromised by the achievement of drug resistance. 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. It has been described that the tumor's ability to survive, grow and metastasize is conditioned by its genetic and phenotypic heterogeneity [3]

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