Gene and protein expression of mTOR and LC3 in hepatocellular carcinoma, colorectal liver metastasis and "normal" liver tissues

Abstract

The physiological role of autophagy in the progression of liver diseases is still debated. To understand the clinical relevance of autophagy in primary e secondary hepatic tumors, we analyzed the expression of mTOR (mammalian target of rapamycin), a key regulator of autophagy; Raptor (regulatory-associated protein of mTOR); ULK1 (Unc-51 like kinase 1) determinant in the autophagy initiation; LC3 (microtubule-associated protein 1A/1B-light chain 3), a specific marker of autophagosomes; and p62, a selective autophagy receptor. Samples from subjects with chronic hepatitis (n.58), cirrhosis (n.12), hepatocellular carcinoma (HCC, n.56), metastases (n.48) from colorectal cancer and hyperplasia or gallbladder stones (n.7), the latter considered as controls, were examined. Gene expression analysis was carried out in n.213 tissues by absolute q-PCR, while protein expression by Western Blot in n.191 lysates, including tumoral, surrounding tumoral and normal tissues. Nonparametric statistical tests were used for comparing expression levels in the above-mentioned groups. Subgroup analysis was performed considering viral infection and chemotherapy treatment. The mTOR transcriptional level was significantly lower in metastases compared to HCC (P = 0.0001). p-mTOR(Ser2448) and LC3II/LC3I protein levels were significantly higher in metastases compared to HCC (P = 0.008 and P<0.0001, respectively). ULK(Ser757) levels were significantly higher in HCC compared to metastases (P = 0.0002) while the HCV- and HBV- related HCC showed the highest p62 levels. Chemotherapy induced a down-regulation of the p-mTOR(Ser2448) in metastases and in non-tumor surrounding tissues in treated patients compared to untreated (P = 0.001 and P = 0.005, respectively). Conclusions: the different expression of proteins considered, owning their interaction and diverse tissue microenvironment, indicate an impairment of the autophagy flux in primary liver tumors that is critical for the promotion of tumorigenesis process and a coexistence of autophagy inhibition and activation mechanisms in secondary liver tumors. Differences in mTOR and LC3 transcripts emerged in tumor-free tissues, therefore particular attention should be considered in selecting the control group.

Fig 1

Gene expression analysis of mTOR/β-actin (A,C,E,G) and LC3/β-actin (B,D,F,H) ratios by quantitative absolute Real-Time PCR using SYBR Green. The unknown mTOR, LC3 and β-actin mRNA amounts in the samples were extrapolated by the respective standard curves performed using serial dilution (1:10) from 10⁸ to 10² copies/μl of a reference sample. From each sample, the normalized amount of the mTOR and LC3 was obtained by the ratio of mTOR and LC3 to β-actin, used as housekeeping gene. Data are represented as box plot: Box is drawn between the first and third quartiles and the line marks the median, the bar the min to max value. The results of mTOR/β-actin ratios and LC3/β-actin ratios were reported considering: A, B) all liver tissues in relation to the pathology vs controls; C, D) tissues separated according to the presence (Virus) or absence (Virus-) of viral infection; E, F) tissues with HCV-related disease; G, H) tissues with HBV-related disease. CTRL: Control group, tissues obtained from cholecystectomy and surrounding hyperplasia. CH: Chronic hepatitis; CIRR: Cirrhosis; PHCC: Cirrhotic tissues surrounding hepatocellular carcinoma; HCC: Hepatocellular carcinoma; HCV: Tissue from hepatitis C virus-related; HBV: Tissue from hepatitis B virus-related; Virus: All tissues with viral infection; Virus-: All tissues without viral infection; NM: Histologically normal liver tissues resected far as possible from the metastatic nodule; PM: Liver tissues surrounding the metastatic nodule; M: Metastatic nodules. P = 0.05:*; P = 0.005: **; P = 0.0001:***; P<0.00001: ****.

Fig 2. mTOR/β-actin and LC3/β-actin controls ratios in metastatic tissues of treated and untreated patients before the surgery.

A) results of mTOR/β-actin gene expression. B) results of LC3/β-actin gene expression. CRTL: Control group. NM NA: Tissues resected far as possible from the metastatic nodule of patients that not received adjuvant chemotherapy (NA) before the surgery; PM NA: Liver tissues surrounding the metastatic nodule of patients that not received adjuvant chemotherapy before the surgery. M NA: Metastatic nodule of patients that not received adjuvant chemotherapy before the surgery. NA: No therapy before the surgery. CT: Adjuvant chemotherapy before the surgery. P = 0.05:*; P = 0.005: **; P = 0.0001:***; P<0.00001: ****.

Fig 3

Western Blot analysis representative bands relative to: p-mTOR(Ser2448), mTOR and β-actin protein expression of patients with primary liver cancer (A) and with liver metastases from CRC (B); p-ULK(Ser757), ULK1 and β-actin protein expression of patients with primary liver cancer (C) and liver metastases from colorectal cancer (D); p-Raptor(Ser792), Raptor and β-actin protein expression of patients with primary liver cancer (E) and liver metastases from colorectal cancer (F); LC3-I, LC3-II and β-actin protein expression of patients with primary liver cancer (G) and liver metastases from colorectal cancer (H); p62 and β-actin protein expression of patients with primary liver cancer (I) and liver metastases from colorectal cancer (L). For p-mTOR(Ser2448), mTOR, p-ULK (Ser757), ULK1, Raptor analysis, given their molecular weight of 289, 140 and 150 KDa respectively, the protein lysate was loaded onto 7.5% SDS-PAGE, onto 10% for p62 KDa and for LC3-I, LC3-II and β-actin analysis, the protein lysate was loaded onto 15% SDS-PAGE, given their molecular weights of 16KDa, 14KDa and 45KDa respectively. PH: Liver tissues surrounding hepatocellular carcinoma; HCC: Hepatocellular carcinoma; NM: Histologically normal liver tissues resected far as possible from the metastatic nodule; PM: Liver tissues surrounding the metastatic nodule; M: Metastatic nodules.

Fig 4

Quantitative Western Blot analysis of p-mTOR/mTOR (A,B,C,D), p-ULK(Ser4757)(E,F,G,H) and p-Raptor(Ser792) (I,L,M,N) ratio. Bands intensity were quantified by optical density using Uvitec Alliance Instrument and Uvitec Alliance software. Values are reported as box plot. The results were reported considering: A, E, I) primary and secondary tumours and in tissues surrounding tumors; B, F, L) tissues separated according to the presence (Virus) or absence (Virus-) of viral infection; C, G, M) tissues with HCV-related and HBV-related disease; D, H, N) metastatic tissues of treated (CT) and untreated (NA) patients before the surgery. PHCC: Cirrhotic tissues surrounding hepatocellular carcinoma; PHCC HCV: Cirrhotic tissues surrounding hepatocellular carcinoma HCV-related. PHCC HBV: Cirrhotic tissues surrounding hepatocellular carcinoma HBV-related. HCC: Hepatocellular carcinoma; HCC HCV: Hepatocellular carcinoma HCV-related. HCC HBV: Hepatocellular carcinoma HBV-related. NM: Histologically normal liver tissues resected far as possible from the metastatic nodule; PM: Liver tissues surrounding the metastatic nodule; M: Metastatic nodules.

Fig 5

Quantitative Western Blot analysis of LC3-II/LC-3I, (A,B,C,D) and p62 (E,F,G,H) ratio. Bands intensity were quantified by optical density using Uvitec Alliance Instrument and Uvitec Alliance software. Values are reported as box plot. The results were reported considering: A, E) primary and secondary tumours and in tissues surrounding tumours; B, F) tissues separated according to the presence (Virus) or absence (Virus-) of viral infection; C, G) tissues with HCV-related and HBV-related disease; D, H) metastatic tissues of treated (CT) and untreated (NA) patients before the surgery. PHCC: Cirrhotic tissues surrounding hepatocellular carcinoma; PHCC HCV: Cirrhotic tissues surrounding hepatocellular carcinoma HCV-related. PHCC HBV: Cirrhotic tissues surrounding hepatocellular carcinoma HBV-related. HCC: Hepatocellular carcinoma; HCC HCV: Hepatocellular carcinoma HCV-related. HCC HBV: Hepatocellular carcinoma HBV-related. NM: Histologically normal liver tissues resected far as possible from the metastatic nodule; PM: Liver tissues surrounding the metastatic nodule; M: Metastatic nodules.