The WSB1 gene is involved in pancreatic cancer progression

Abstract

Background: Pancreatic cancer cells generate metastases because they can survive the stress imposed by the new environment of the host tissue. To mimic this process, pancreatic cancer cells which are not stressed in standard culture conditions are injected into nude mice. Because they develop xenografts, they should have developed adequate stress response. Characterizing that response might provide new strategies to interfere with pancreatic cancer metastasis.

Methodology/principal findings: In the human pancreatic cancer cell lines Panc-1, Mia-PaCa2, Capan-1, Capan-2 and BxPC3, we used Affymetrix DNA microarrays to compare the expressions of 22.000 genes in vitro and in the corresponding xenografts. We identified 228 genes overexpressed in xenografts and characterized the implication of one of them, WSB1, in the control of apoptosis and cell proliferation. WSB1 generates 3 alternatively spliced transcripts encoding distinct protein isoforms. In xenografts and in human pancreatic tumors, global expression of WSB1 mRNA is modestly increased whereas isoform 3 is strongly overexpressed and isoforms 1 and 2 are down-regulated. Treating Mia-PaCa2 cells with stress-inducing agents induced similar changes. Whereas retrovirus-forced expression of WSB1 isoforms 1 and 2 promoted cell growth and sensitized the cells to gemcitabine- and doxorubicin-induced apoptosis, WSB1 isoform 3 expression reduced cell proliferation and enhanced resistance to apoptosis, showing that stress-induced modulation of WSB1 alternative splicing increases resistance to apoptosis of pancreatic cancer cells.

Conclusions/significance: Data on WSB1 regulation support the hypothesis that activation of stress-response mechanisms helps cancer cells establishing metastases and suggest relevance to cancer development of other genes overexpressed in xenografts.

Figure 1. The three proteic isoforms of wsb1 and their subcellular localization.

A. Schematic representation of WSB1 isoforms 1, 2 and 3. B. WSB1 isoforms were expressed as EGFP or V5 tag fusion proteins and subcellular localization was analyzed by direct fluorescence of after immunocytochemistry using EGFP or V5 antibodies respectively.

Figure 2. Splicing is in favor of isoform 3 in mice xenografts, human pancreatic tumors and after stress.

Expression of WSB1 isoforms 1+2 and 3 mRNAs was assessed by qRT-PCR in pancreatic cancer cell lines and in mice xenografted tumors (A), in human pancreatic cancer samples (B), and in Mia-PaCa2 cells after treatment with stress-inducing agents (C). Values are expressed as the mean±S.D. of combined results from two independent experiments performed in triplicate. (*, p<0.05).

Figure 3. WSB1 enhances cell proliferation.

A. Mia-PaCa2 cells were transfected with a siRNA directed against WSB1 mRNA and expression of WSB1 isoforms 1+2+3, 1+2 and 3 mRNAs was analyzed by qRT-PCR. B. Mia-PaCa2 cells were transfected with the WSB1 siRNA and growth was analyzed by direct cell counting (B), MTT analysis (C) or by BrdU incorporation (D) as described in Material and Methods section. E. Mia-PaCa2 cells were transfected with the WSB1 siRNA and caspase 3 activity was measured in untreated and after treatment with staurosporine or doxorubicin. Values are expressed as the mean±S.D. of combined results from two independent experiments performed in triplicate. (*, p<0.05).

Figure 4. A specific role for isoform 3 which reduces pancreatic cancer cell growth.

A. Mia-PaCa2 cells were transduced with retrovirus expressing WSB1 isoforms 1, 2 or 3 as EGFP fusion protein or EGFP alone as a control and selected with puromycin. Cell growth was measured by direct counting after plating 10⁵ cells in 48-well dishes every day during 4 days. B. After synchronization with 24 h serum-starvation, cells were incubated in serum containing medium for 24 h, stained with propidium iodide and cell cycle was analysed with a flow cytometer. C. S/G1 ratio. Values are expressed as the mean±S.D. of combined results from two independent experiments performed in triplicate (A) or trice in triplicate (B and C) (*, p<0.05).

Figure 5. WSB1 isoform 3 reduces susceptibility to stress-induced cell death.

Mia-PaCa2 cells were transduced with retrovirus expressing WSB1 isoforms 1, 2 or 3 as EGFP fusion protein or EGFP as control and selected with puromycin. Cells were treated with gemcitabine (100 and 150 µM) for 48 hours or with doxorubicin (150 and 350 µM) for 6 hours. Cell viability was measured by MTT. Values are expressed as the mean±S.D. of combined results from two independent experiments performed in triplicate. (*, p<0.05).