Hsp70 exerts oncogenic activity in the Apc mutant Min mouse model

Abstract

Colorectal cancer (CRC) develops from colonic epithelial cells that lose expression of key tumor suppressor genes and/or gain expression of proproliferative and antiapoptotic genes like heat shock protein 70 (Hsp70). Heat shock protein 70 is overexpressed in CRC, but it is not known whether this is in response to the proteotoxic stress induced by transformation, or if it contributes to the process of transformation itself. Here, using the Apc (Min/+) mouse model of CRC, we show that Hsp70 regulates mitogenic signaling in intestinal epithelial cells through stabilization of proteins involved in the receptor tyrosine kinase (RTK) and WNT signaling pathways. Loss of Hsp70 reduced tumor size with decreased proliferation and increased tumor cell death. Hsp70 loss also led to decreased expression of ErbB2, Akt, ERK and β-catenin along with decreased β-catenin transcriptional activity as measured by c-myc and axin2 expression. Upregulation of RTK or WNT signals are frequent oncogenic events in CRC and many other cancers. Thus, in addition to the role of Hsp70 in cell-survival after transformation, Hsp70 stabilization of β-catenin, Akt, ERK and ErbB2 are predicted to contribute to transformation. This has important implications not only for understanding the pathophysiology of these cancers, but also for treatment since anti-EGFR antibodies are in clinical use for CRC and EGFR is a major ErbB2 heterodimeric partner. Targeting Hsp70, therefore, might provide an alternative or complementary strategy for achieving better outcomes for CRC and other related cancer types.

Figure 1.

Hsp70 expression is increased in human CRC and a mouse model of intestinal tumorigenesis and loss of Hsp70 results in fewer and smaller intestinal tumors in Apc^Min/+ mice. (A) Immunostaining for Hsp70 (brown) in formalin-fixed, paraffin-embedded (FFPE) normal human colon, a colonic polyp from a patient with familial adenomatous polyposis and sporadic human colonic adenocarcinoma. (B) Immunoblotting for Hsp70 in lysates of normal intestinal mucosa of Apc^+/+ and Apc^Min/+ mice and tumors isolated from the small intestine of Apc^Min/+ mice. Blots are representative of normal mucosa and tumors from individual Apc^+/+ (n = 1) and Apc^Min/+ (n = 5) mice. (C) Survival curve of Apc^Min/+Hsp70^+/+ and Apc^Min/+Hsp70^−/− mice. All mice were on C57Bl6/J background and included 10 mice in each group. (D) Number of tumors per mouse (mean ± S.E.M) in Apc^Min/+Hsp70^+/+ mice (n = 18) and Apc^Min/+Hsp70^−/− (n = 29) mice. Mice were sacrificed at 6 months of age. Top panel: representative intestines from indicated mice. (E) Tumor size distribution in 6 month old Apc^Min/+Hsp70^+/+ (n = 18) and Apc^Min/+Hsp70^−/− (n = 29) reported as mean ± S.E.M. ***P < 0.005; ****P < 0.001, compared to Apc^Min/+Hsp70^+/+ mice.

Figure 2.

Hsp70 regulates proliferation and cell death. (A) Apoptosis assessed by TUNEL staining (brown) in tumors from FFPE Apc^Min/+Hsp70^+/+ (n = 5) and Apc^Min/+Hsp70^−/− (n = 5) tumors. (B) PARP cleavage assessed by Western blotting in lysates from Apc^Min/+Hsp70^+/+ (n = 6) and Apc^Min/+Hsp70^−/− (n = 7) tumors. (C) Proliferation as assessed by BrdU incorporation (brown) after 2h labeling in vivo in Apc^Min/+Hsp70^+/+ (n = 3) and Apc^Min/+Hsp70^−/− (n = 3) tumors (mean ± S.E.M), ***P < 0.05 compared to Apc^Min/+Hsp70^+/+. (D) Hsp70^+/+ and Hsp70^−/− MEFs were plated in 96-well plates at 5×10⁴ cells/well in two independent experiments and the number of viable cells quantified by WST-1 assay. ****P < 0.001, compared to Hsp70^+/+ MEFs.

Figure 3.

β-catenin transcriptional activity is decreased in Hsp70^−/− cells. (A) Immunostaining for β-catenin (brown) in FFPE Apc^Min/+Hsp70^+/+ (n = 10) and Apc^Min/+Hsp70^−/− (n = 6) tumors. Nuclear β-catenin was quantified by determining the number of positive nuclei per 100 cells in images of immunostained cells (mean ± S.E.M; P < 0.05 compared to Apc^Min/+Hsp70^+/+). (B) Human colon cancer cells (Caco-2 in left panel and HCT116 middle panel) were transfected with Hsp70 siRNA or scrambled (control) oligonucleotides for 24h and then transfected with β-catenin/Tcf TOPflash reporter or negative control FOPflash reporter for 24h with values normalized using pRL-TK. Mouse embryonic fibroblasts (right panel) deficient in Hsp70 were transfected with TOPflash or FOPflash reporters. Lysates were assayed for luciferase activity and Hsp70 and β-actin protein levels to verify knockdown in the colon cancer cells (P < 0.05 compared to scrambled siRNA transfected cells). Normalized luciferase activity is reported as β-catenin activation. (C) Axin2 mRNA levels were measured by qRT-PCR on samples from normal small intestine of Apc^+/+Hsp70^+/+ mice (n = 2) and tumors from Apc^Min/+Hsp70^+/+ (n = 3) and Apc^Min/+Hsp70^−/− (n = 2) mice. *P < 0.05. (D) c-Myc as assessed by Western blotting in lysates of tumors from Apc^Min/+Hsp70^+/+ (n = 7) and Apc^Min/+Hsp70^−/− (n = 7) mice.

Figure 4.

Hsp70 controls β-catenin protein stability in tumor cells. (A) β-catenin levels were measured by Western blotting in lysates from Apc^Min/+Hsp70^+/+ (n = 7) and Apc^Min/+Hsp70^−/− (n = 7) tumors. Each lane represents lysates from a different tumor. Densitometry was used to quantify protein expression. (***P < 0.005 compared to Apc^Min/+Hsp70^+/+). (B) Cell lysates from Caco2 or HCT116 cells were immunoprecipitated with anti-Hsp70 or anti-β-catenin antibodies or IgG alone (negative control) and then probed for Hsp70 or β-catenin. (C) Ubiquitinated β-catenin. β-catenin was immunoprecipitated from tumor lysates and the immunoprecipitates were probed for ubiquitin. Quantification of ubiquitin by densitometry is shown in the right panel. *P < 0.05 compared to Apc^Min/+Hsp70^+/+. (D) Cells isolated from tumors in Apc^Min/+Hsp70^+/+ and Apc^Min/+Hsp70^−/− mice were treated with a vehicle control or 20 μM MG132 for 2h then lysed and immunoblotted for Hsp70, β-catenin and actin.

Figure 5.

The Ras-Raf-MAPK and PI3K-AKT-mTor signaling cascades are dependent upon Hsp70. (A) Immunoblotting for activated proteins involved in the Ras-Raf-MAPK (ERK and P38) and PI3K-AKT-mTor (AKT) signaling cascades in lysates from Apc^Min/+Hsp70^+/+ (n = 7) and Apc^Min/+Hsp70^−/− (n = 7) tumors. (B) Hsp70, ERK or AKT were immunoprecipitated from tumor lysates and the immunoprecipitates were probed for the corresponding proteins. (C) Caco-2 cells were transfected with Top Flash or Fop Flash (negative control) for 24h, then treated with PD98059 (PD) for 2h and lysates were assayed for phospho-ERK (p-ERK) and total β-catenin as well as luciferase activity. Normalized luciferase activity is reported as β-catenin activation. *P < 0.05, compared to vehicle-treated cells.

Figure 6.

Hsp70 coassociates with ErbB2 and regulates ErbB2 protein levels. (A) Quantitative RT-PCR for ErbB2 mRNA expression in tumors from Apc^Min/+Hsp70^+/+ (n = 3) and Apc^Min/+Hsp70^−/− (n = 3) mice. (B) Total and phosphorylated ErbB2 as assessed by Western blotting in lysates from Apc^Min/+Hsp70^+/+ (n = 4) and Apc^Min/+Hsp70^−/− (n = 4) tumors. (C) Co-association of Hsp70 and ErbB2 in lysates of Apc^Min/+Hsp70^+/+ tumors (n = 6). Tumor lysates were immunoprecipitated with Hsp70 or ErbB2 and the IP’s resolved by SDS-PAGE. The conjugate member (ErbB2 for Hsp70 IP and Hsp70 for ErbB2 IP) were probed by Western blotting. (D) Ubiquitinated-ErbB2. ErbB2 was immunoprecipitated and IPs probed for ubiquitin in Apc^Min/+Hsp70^+/+ (n = 6) and Apc^Min/+Hsp70^−/− (n = 6) tumors. The amount of lysate used for immunoprecipitation was adjusted so that levels of ErbB2 were comparable between Apc^Min/+Hsp70^+/+ and Apc^Min/+Hsp70^−/− samples.