Heat shock protein 47 promotes tumor survival and therapy resistance by modulating AKT signaling via PHLPP1 in colorectal cancer

Abstract

Objective: Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that facilitates collagen maturation. Its role in cancer remains largely unknown. In this study, we investigated the roles of HSP47 in colorectal cancer (CRC) and therapy resistance. Methods: Expression of HSP47 in CRC tissues was examined (1) in paired human CRC/adjacent normal tissues, using real time quantitative reverse transcription polymerase chain reaction (qRT-PCR), The Cancer Genome Atlas (TCGA) database, and 22 independent microarray databases (curated CRC). In vitro studies on several CRC cell lines (HCT116, RKO and CCL228) with modulated HSP47 expression were conducted to assess cell viability and apoptosis (TUNEL assay and caspase-3/-7) during exposure to chemotherapy. AKT signaling and co-immunoprecipitation studies were performed to examine HSP47 and PHLPP1 interaction. In vivo studies using tumor xenografts were conducted to assess the effects of HSP47 modulation on tumor growth and therapy response. Results: HSP47 was upregulated in CRC and was associated with poor prognosis in individuals with CRC. In vitro, HSP47 overexpression supported the survival of CRC cells, whereas its knockdown sensitized cells to 5-fluorouracil (5-FU). HSP47 promoted survival by inhibiting apoptosis, enhancing AKT phosphorylation, and decreasing expression of the AKT-specific phosphatase PHLPP1 when cells were exposed to chemotherapy. These effects were partly results of the interaction between HSP47 and PHLPP1, which decreased PHLPP1 stability and led to more persistent AKT activity. In vivo, HSP47 supported tumor growth despite 5-FU treatment. Conclusions: HSP47 supports the growth of CRC tumors and suppresses the efficacy of chemotherapy via modulation of AKT signaling.

Keywords: AKT; HSP47; PHLPP1; colorectal cancer; resistanc.

Figure 1

High HSP47 expression in patients with colorectal cancer (CRC) is associated with poor clinical outcomes. (A) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of HSP47 mRNA expression in paired tumor and adjacent normal tissues collected by laser microdissection from patients with CRC (n = 9 pairs) (P < 0.01). (B) HSP47 mRNA expression in 644 patients with CRC compared with 51 control tissues in a TCGA cohort. (C) HSP47 mRNA expression in 3,296 patients with CRC compared with 76 control tissues from 22 microarray databases (curated CRC Data). Kaplan–Meier survival analysis of the CRC patients in the (D) TCGA and (E) curated CRC Data cohorts. The patients were divided into high- and low-expression groups on the basis of the cutoff value derived from the receiver operating characteristic (ROC) analysis. **P < 0.01; ***P < 0.001.

Figure 2

HSP47 expression promotes drug resistance in colorectal cancer (CRC) cells exposed to chemotherapy. (A) Representative image of Western blot analysis of HSP47 protein expression in various human CRC cell lines. (B) HCT116, (C) RKO, and (D) CCL228 CRC cell lines were transiently transduced with HSP47-expression vector and then exposed to 5-FU. MTS cell viability assays of (E) CCL228 cells exposed to various concentrations of 5-FU after HSP47 transient knockdown. MTS assays of (F) HSP47-overexpressing (HCT116/HSP47_st) and (G) HSP47-knockdown (RKO/si-HSP47_st) stable cell lines exposed to 5-FU. HSP47 mRNA and protein expression in (H) RKO and (I) HCT116 resistant cell lines were determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis, respectively. Data represent mean ± SEM, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 3

HSP47 inhibits apoptosis in colorectal cancer (CRC) cells exposed to chemotherapy. (A) HCT116/HSP47_st cells were visualized by fluorescence microscopy (200×) 48 h after 50 μM 5-FU treatment. TUNEL-positive nuclei are shown in green, and total nuclei stained with Hoechst 33258 are shown in blue. (B) Quantitative analysis (percentage of TUNEL positive cells vs. total) was performed for randomly selected fields (n = 10). Representative Western blot images of caspase-3 and -7 cleavage of (C) HCT116/HSP47_st, (D) RKO/si-HSP47_st, and their corresponding control cell lines after treatment with 50 μM 5-FU. Data represent mean ± SEM. **P < 0.01; ***P < 0.001.

Figure 4

HSP47 promotes AKT activation and decreases PHLPP1 expression in colorectal cancer (CRC) cells exposed to 5-FU. Representative immunoblotting images of (A) HCT116/HSP47_st, (B) RKO/si-HSP47_st, and their corresponding control cell lines treated with 50 μM 5-FU. The immunoblotting images of (C, D) phospho-Akt (S473) and (E, F) PHLPP1 were quantified in Image J software (n = 3). Data represent mean ± SEM. *P < 0.05.

Figure 5

HSP47 interacts with PHLPP1 and decreases its protein stability. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of PHLPP1 mRNA expression levels in (A) HCT116/HSP47_st, (B) RKO/si-HSP47_st, and their corresponding control cell lines treated with 50 μM 5-FU (n = 3). (C) Representative Western blot images of RKO/si-HSP47_st and its control cell line treated with 100 μg/mL cycloheximide (CHX). Cells were collected at the indicated time points and subjected to Western blot analysis. The immunoblotting images were quantified in Image J software (n = 3). (D) RKO cells were double stained with the mouse monoclonal antibody against PHLPP1 (green) and rabbit monoclonal antibody against HSP47 (red), then subjected to confocal immunofluorescence analysis. Colocalization between PHLPP1 and HSP47 is indicated by arrows. (E) Co-immunoprecipitation of HSP47 with PHLPP1 in parental RKO cells. Scale bar = 5 μm. Data represent mean ± SEM. *P < 0.05.

Figure 6

HSP47-overexpressing colorectal cancer (CRC) tumors do not respond to chemotherapy. (A) In vivo growth of subcutaneous mouse xenografted tumors of HCT116/HSP47_st and the control cell line treated with either vehicle (Vh) or 5-FU (n = 4 per group). (B) Western blot analysis of proteins extracted from tumor tissues collected from vehicle-treated mice. (C) FFPE xenograft tissues were double stained with mouse monoclonal antibody against PHLPP1 (red) and rabbit monoclonal antibody against HSP47 (green), then subjected to confocal immunofluorescence analysis. Arrows indicate colocalization between PHLPP1 and HSP47. Scale bar = 20 μm. Data represent mean ± SEM. ***P < 0.001.

Figure 7

Schematic diagram depicting how HSP47 decreases the protein stability of PHLPP1 and promotes Akt signaling and chemoresistance in colorectal cancer (CRC). The ligand-mediated activation of receptor tyrosine kinase (RTK) promotes recruitment of phosphoinositide 3-kinase (PI3K) to the plasma membrane via its regulatory domain (p85), thus triggering activation of PI3K and conversion, via the catalytic domain (p110), of phosphatidylinositol-3,4-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-tris-phosphate (PIP3). Akt binds the PIP3 at the plasma membrane via its PH domain, thus allowing PDK1 to access and phosphorylate Thr308 in the kinase domain, and leading to partial Akt activation. Phosphorylation of Akt at Ser473 in the regulatory domain by mTORC2 stimulates full Akt activity. Dephosphorylation of Thr308 by PP2A, dephosphorylation of Ser473 by PHLPP1, and the conversion of PIP3 to PIP2 by phosphatase and tensin homolog (PTEN) antagonize Akt signaling. We propose that HSP47 promotes cell survival and chemoresistance by interacting with PHLPP1. The binding of HSP47 facilitates PHLPP1 protein degradation, thereby maintaining and supporting the full activity of Akt kinase required for cell survival in CRC cells receiving chemotherapy.