Identification of genes involved in resistance to interferon-alpha in cutaneous T-cell lymphoma

Abstract

Interferon-alpha therapy has been shown to be active in the treatment of mycosis fungoides although the individual response to this therapy is unpredictable and dependent on essentially unknown factors. In an effort to better understand the molecular mechanisms of interferon-alpha resistance we have developed an interferon-alpha resistant variant from a sensitive cutaneous T-cell lymphoma cell line. We have performed expression analysis to detect genes differentially expressed between both variants using a cDNA microarray including 6386 cancer-implicated genes. The experiments showed that resistance to interferon-alpha is consistently associated with changes in the expression of a set of 39 genes, involved in signal transduction, apoptosis, transcription regulation, and cell growth. Additional studies performed confirm that STAT1 and STAT3 expression and interferon-alpha induction and activation are not altered between both variants. The gene MAL, highly overexpressed by resistant cells, was also found to be expressed by tumoral cells in a series of cutaneous T-cell lymphoma patients treated with interferon-alpha and/or photochemotherapy. MAL expression was associated with longer time to complete remission. Time-course experiments of the sensitive and resistant cells showed a differential expression of a subset of genes involved in interferon-response (1 to 4 hours), cell growth and apoptosis (24 to 48 hours.), and signal transduction.

Figure 1.

Comparison of the IFN-α sensitivity of huT78S and huT78R. A: The parental huT78 (huT78S) was tested for its sensitivity to IFN-α by culturing cells throughout 96 hours in concentrations of between 0 U/ml IFN-α and 1,000,000 U/ml IFN-α. The viability of cells was determined every 24 hours by trypan blue exclusion. These cells are sensitive to IFN-α at concentrations as low as 100 U/ml. The IC50 for this cell line at 96 hours was calculated to be between 100 U/ml and 1000 U/ml. B: The huT78R cell line produced was tested for its IFN-α sensitivity after 1 month of growth in the absence of IFN-α. As can be seen above this variant strain is extremely resistant to IFN-α even at concentrations of IFN-α of 1,000,000 U/ml.

Figure 2.

Basal expression of STAT1 and STAT3 in huT78S and huT78R and the changes induced in the expression of these genes by IFN-α (5000 U/ml) after 3 and 24 hours. A: Quantitative PCR results showing that the basal levels of STAT1 and STAT3 are not significantly different between huT78S and huT78R variants. B: Cells from huT78S and huT78R were harvested before commencement of treatment and 3 and 24 hours after commencement of treatment of the cultures with 5000 U/ml IFN-α. Quantitative PCR for STAT1 showed a rapid and significant increase in STAT1 mRNA levels in the case of both huT78S and huT78R. C: STAT3 was also studied under the same conditions. In the case of huT78S there is a slight up-regulation of STAT3 in response to IFN-α. There is no significant change in expression observed in the huT78R variant.

Figure 3.

STAT1 and STAT3 expression and activation by IFN-α in huT78S and huT78R. Cells from huT78S and huT78R were collected by cytospin and fixed in acetone before commencement of treatment and 3 and 24 hours after commencement of treatment of the cultures with 5000 U/ml IFN-α (0 and 24 hours only shown here). Cytospins were stained with both STAT1 and STAT3 antibodies. In huT78S (A) and huT78R (B) initially STAT1 protein is found in the cytoplasm. At 24 hours STAT1 has been activated with protein clearly visible and concentrated in the nucleus. In huT78S (C) and huT78R (D) STAT3 is also initially located in the cytoplasm. The protein is activated and translocated to the nucleus after 24 hours.

Figure 4.

Comparison of the results achieved using microarray analysis and quantitative PCR between the two variants. To validate the microarray result, quantitative PCR was performed for eight genes using TaqMan probes (five genes; MAL, SAMSN1, BAG3, STAT1, and STAT3) or SYBR green (three genes; TNFSF7, CAV1, and P2Y5). The genes studied included: ▨, genes up-regulated huT78R (MAL, SAMSN1), ▥, genes whose expression was unchanged between the two variants (STAT1, STAT3), and ▧, genes down-regulated in huT78R (BAG3, TNFSF7, CAV1, P2Y5). For all eight genes studied the microarray results were confirmed and in most cases the results obtained from both techniques were extremely similar and no differences observed were significant.

Figure 5.

Immunohistochemical study of MAL protein in paraffin sections of MF tumoral tissue from patients treated with IFN-α. A and B: A case of MF in a patient in which MAL is not detected. B and D: A case of a MF in a patient positive for MAL and who shows a slow response in the case of IFN-α treatment.

Figure 6.

A: Cluster of IFN-α early response genes (1 and 4 hours) in huT78S and huT78R. CI: Genes down-regulated in huT78R, expression in huT78S does not change. CII-A: Genes up-regulated in huT78S, expression in huT78R does not change or is up-regulated but delayed. CIII: Genes up-regulated in huT78R, expression change in huT78S variable (unchanged, down-regulated or up-regulated but delayed). CIV: Genes down-regulated in huT78S and either up-regulated in huT78R and/or down-regulated but delayed. B: Cluster of IFN-α late response genes (24 and 48 hours) in huT78S and huT78R. CI: Genes up-regulated in the huT78R, expression in huT78S does not change or down-regulation is observed. CII: Genes down-regulated in huT78S, expression in huT78R does not change. CIII: Genes down-regulated in huT78R, expression in huT78S does not change. CIV: Genes up-regulated in huT78S and expression does not change in huT78R.

Figure 7.

Apoptotic and signal transduction pathways disrupted in the IFN-α resistant cell line detected by microarray analysis. Expression of several genes acting in concert to regulate the balance between proliferation and programmed cell death is altered in the resistance variant (hut78R) as compared to the sensitive variant (hut78S). Up-regulated (red text label) and down-regulated genes (green text label) in huT78R versus huT78S are noted. Changes in the expression of several genes could possibly be the cause of the resistance phenotype (as the up-regulation of TNFSF7 and MAL and the down-regulation of SHD2H1A and CAV1) although some others may be a consequence.