MUC1c Regulates Cell Survival in Pancreatic Cancer by Preventing Lysosomal Permeabilization

<div><h3>Background</h3><p>MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells including pancreatic cancer. The cytosolic end of MUC1 (MUC1-c) is extensively involved in a number of signaling pathways. MUC1-c is reported to inhibit apoptosis in a number of cancer cells, but the mechanism of inhibition is unclear.</p> <h3>Method</h3><p>Expression of MUC1-c was studied in the pancreatic cancer cell line MIAPaCa-2 at the RNA level by using qRTPCR and at the protein level by Western blotting. MUC1-c expression was inhibited either by siRNA or by a specific peptide inhibitor, GO-201. Effect of MUC1-c inhibition on viability and proliferation and lysosomal permeabilization were studied. Association of MUC1-c with HSP70 was detected by co-immunoprecipitation of MUC1-c and HSP70. Localization of MUC1-c in cellular organelles was monitored by immunofluorescence and with immuno- blotting by MUC1-c antibody after subcellular fractionation.</p> <h3>Results</h3><p>Inhibition of MUC1-c by an inhibitor (GO-201) or siRNA resulted in reduced viability and reduced proliferation of pancreatic cancer cells. Furthermore, GO-201, the peptide inhibitor of MUC1-c, was effective in reducing tumor burden in pancreatic cancer mouse model. MUC1-c was also found to be associated with HSP70 in the cytosol, although a significant amount of MUC1 was also seen to be present in the lysosomes. Inhibition of MUC1 expression or activity showed an enhanced Cathepsin B activity in the cytosol, indicating lysosomal permeabilization. Therefore this study indicates that MUC1-c interacted with HSP70 in the cytosol of pancreatic cancer cells and localized to the lysosomes in these cells. Further, our results showed that MUC1-c protects pancreatic cancer cells from cell death by stabilizing lysosomes and preventing release of Cathepsin B in the cytosol.</p> </div

Evaluation of metastasis in the mouse model for pancreatic cancer.

<p>Evaluation of metastasis in the mouse model for pancreatic cancer.</p

Inhibition of MUC1 expression or activity results in reduced proliferation and cell viability.

<p>MUC1 expression was inhibited by siRNA in MIAPaCa-2 (A) Lanes 1–3 show control MIAPaCa-2, MIAPaCa-2 transfected with non-specific siRNA and MUC1 siRNA respectively. Both proliferation (B) and viability (C) were reduced with decreased expression of MUC1 in MIAPaCa-2 cells. On treatment with MUC1-C activity inhibitor GO-201, which inhibits the signaling activity of this protein, no change was seen in expression levels of MUC1 in MIAPaCa-2 cells (D). Lane 1 was untreated MIAPaCa-2 cells and lane 2 was MIAPaCa-2 treated with GO-201. Proliferation (E) and viability (F) were seen to be decreased. Similarly, in another cell line, AsPC-1, transfection with siRNA showed decreased protein levels (G) where Lane 1 is control AsPC-1, Lane 2: non-specific siRNA transfected AsPC-1 and Lane 3 is MUC1 siRNA-transfected AsPC1. Both proliferation (H) and viability (I) were reduced. On treatment with the inhibitor GO-201, there was no change in protein levels (J): Lane 1 was untreated AsPC-1 cells and lane 2 was AsPC-1 treated with GO-201. Proliferation (K) and viability (L) were seen to be reduced. Data are expressed as mean+/−SEM of 3 independent experiments. *<i>P</i><.05 (<i>t</i> test) as compared with controls.</p

MUC1 is expressed in most pancreatic cancer cell lines and mouse models.

<p>mRNA expression levels of MUC1 in several pancreatic cancer cell lines relative to non-tumorigenic HPDEC (A). Data are expressed as mean+/−SEM of 3 independent experiments. *<i>P</i><.05 (<i>t</i> test) as compared with controls. Protein expression levels of MUC1-c in different pancreatic cancer cell line and non-tumorigenic HPDEC (B).</p

MUC1-c associates with HSP70 and localizes in the lysosomes.

<p>Immunoprecipitation with MUC1 and HSP70 showed the two proteins to be associated (A). Lanes 1, 2 and 3 show total protein levels, HSP70 and MUC1 in the flowthrough and the two proteins immunoprecipitated by HSP70 antibody respectively. Lanes 4 and 5 show flowthrough and immunoprecipitates by MUC1 antibody. MUC1-c was found to be localized to the lysosomes in MIAPaCa-2 cells, though almost equal amounts of MUC1-c were also cytosolic (B). Lamp2, a resident lysosomal protein, and actin, a predominantly cytosolic protein, were used as fractionation markers. Lane 1 is the cytosolic fraction. Lane 2 is a crude lysosomal fraction, which is further enriched in Lane 3. HSP70, though associated with MUC1, was seen to be present predominantly in the cytosol. Immunofluorescence confirmed co-localization of MUC1-c and Lamp2 in lysosomes (C).</p