Chaperone-Mediated Autophagy in the Liver: Good or Bad?

Abstract

Hepatitis C virus (HCV) infection triggers autophagy processes, which help clear out the dysfunctional viral and cellular components that would otherwise inhibit the virus replication. Increased cellular autophagy may kill the infected cell and terminate the infection without proper regulation. The mechanism of autophagy regulation during liver disease progression in HCV infection is unclear. The autophagy research has gained a lot of attention recently since autophagy impairment is associated with the development of hepatocellular carcinoma (HCC). Macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) are three autophagy processes involved in the lysosomal degradation and extracellular release of cytosolic cargoes under excessive stress. Autophagy processes compensate for each other during extreme endoplasmic reticulum (ER) stress to promote host and microbe survival as well as HCC development in the highly stressed microenvironment of the cirrhotic liver. This review describes the molecular details of how excessive cellular stress generated during HCV infection activates CMA to improve cell survival. The pathological implications of stress-related CMA activation resulting in the loss of hepatic innate immunity and tumor suppressors, which are most often observed among cirrhotic patients with HCC, are discussed. The oncogenic cell programming through autophagy regulation initiated by a cytoplasmic virus may facilitate our understanding of HCC mechanisms related to non-viral etiologies and metabolic conditions such as uncontrolled type II diabetes. We propose that a better understanding of how excessive cellular stress leads to cancer through autophagy modulation may allow therapeutic development and early detection of HCC.

Keywords: chaperone-mediated autophagy (CMA); endoplasmic reticulum stress (ER stress); hepatitis C virus (HCV); hepatocellular carcinoma (HCC); interferon alpha receptor 1 (IFNAR1); nuclear factor erythroid 2-related factor 2 (NRF2).

Figure 1

Hepatic adaptive response to multifaceted stress generated during chronic hepatitis C virus (HCV) infection is called the integrated stress response (ISR). Shown is the summary of multifaceted stress response generated during chronic HCV infection that is associated with the risk of liver disease progression. In addition to the direct virus-induced ER stress/UPR gene expression, many different cellular stress signals are induced in HCV-infected cells due to a shift in host cell metabolism. There are four different stress kinases participate in the generation of multifaceted stress response. Stress signals during HCV infection activate PKR, GCN2, PERK, and HRI kinases that stimulate phosphorylation of eIF2α, the core element of the stress response, which inhibits cellular translation. Under normal conditions with low levels of phosphorylated eIF2α promotes cap-dependent translation. During the ISR, cellular translation is attenuated due to increased eIF2α phosphorylation, which supports the translation of specific gene (ATF4) needed for cell survival. The PERK-eIF2α-ATF4 activation has been associated with autophagy induction and cell survival. If the stress becomes severe this can also activate NRF2 transcription and chaperone-mediated autophagy (CMA) activation.

Figure 2

Shown is autophagy in the healthy liver. Hepatic autophagy supplies amino acids, glucose, and free fatty acids to meet the energy demand. Autophagy is activated by various stress signals derived from the ER that senses low energy state by MTOR1 and AMPK. The frequent target of these signaling pathways is the ULK complex consisting of ULK1/2, ATG13, RB1 inducible coiled-coil protein 1 (FIP200) and ATG101). This triggers the nucleation of the phagophore by phosphorylating components of the class III P13K (P13KC3) complex 1 [consisting of class III P13K, vacuolar protein sorting 34 (VPS34), beclin 1, ATG14, activating molecule in beclin 1-regulated autophagy protein 1(AMBRA1) and general vesicular transport factor p115], which activates local phosphatidylinositol-3-phosphate (PI3P) at ER membrane called omegasome. PI3P then recruits its effector proteins WD repeat domain phosphoinositide-interacting proteins (WIPIs) and Zinc-finger FYVE domain-containing protein 1 (DFCP1) to the omegasome. WIP12 binds to ATG16L directly, therefore, recruiting the ATG12-ATG5-ATG16L complex. This facilitates the ATG3-mediated conjugation of microtubule-associated protein 1 light chain 3 alpha (LC3) proteins. The conjugation reaction, which LC3-I is converted to LC3-II, is the characteristic features of autophagic membranes. Sealing of the autophagosomal membrane generates autophagosomes, which matures after fusion with endosome and then fuses with the lysosome. The acidic hydrolases in the lysosome degrade the autophagic cargo and nutrients are released back to the cytoplasm for reuse. The metabolites such as amino acids, sugars, and lipids are then released into the cytoplasm for the synthesis of new macromolecules.

Figure 3

Shown is an illustration demonstrating through which excessive cellular stress at the ER activates other two forms of autophagy (microautophagy or CMA) as an efficient cellular compensatory process to reduce cellular stress and improve cell survival. CMA requires a specific cytosolic protein containing a KFERQ-like pentapeptide sequence that is recognized by HSC70 and subsequently translocated into the lysosomal lumen via interaction with LAMP2A for degradation. Microautophagy involves direct incorporation of cytoplasmic materials into endosomes or lysosomes for either degradation or export outside to reduce cellular stress. Microautophagy is also involved in the exosome secretion of cytoplasmic cargo to reduce cellular stress.

Figure 4

Shown are the steps involved in CMA. Cellular stress activates NRF2-related CMA, which is one of the mechanisms to improve cell survival. NRF2 induces transcription of HSC70 and LAMP2A, which are two critical molecules involved in CMA activity. CMA protein substrate containing KFERQ motifs binds to HSC70 and other chaperones. The complex binds to LAMP2A expressed on the surface of the lysosome. This promotes LAMP2A oligomerization that leads to substrate unfolding, translocation, and lysosomal degradation. GFAP favors the LAMP2A oligomerization as phosphorylation of GFAP by AKT prevents LAMP2A oligomerization and results in inhibition of CMA. Lysosomal mTORC2-mediated AKT phosphorylation inhibits CMA. PHLPP1 upregulates CMA activity through dephosphorylating AKT.

Figure 5

Shown is the immunohistochemical staining demonstrating remarkable differences in the pattern of LAMP2A staining between hepatocellular carcinoma (HCC) and surrounding non-tumorous hepatocytes in the cirrhotic liver. The LAMP2A staining in the HCC tumor is mostly cytoplasmic probably due to lysosome amplification or LAMP2A transcription indicating increased CMA activity. Arrows shows the bile canalicular accentuation in the non-tumorous cirrhotic liver.

Figure 6

Shown is the immunohistochemical staining pattern of p62 and LAMP2A in HCC and surrounding non-tumor areas of the cirrhotic livers. (A). The appearance of p62 is significantly higher in HCC as compared to the adjacent non-tumorous cirrhotic liver, suggesting that HCC has impaired autophagy response. High-power micrograph shows cytoplasmic accumulation of p62 only in the HCC, but not the surrounding non-tumorous hepatocytes in the cirrhotic liver. (B). LAMP2A staining of HCC tumor nodule developed in cirrhotic areas. The LAMP2A staining is different between HCC tumors in the center and hepatocytes in surrounding non-tumorous cirrhotic areas. High-power micrograph clearly shows cytoplasmic LAMP2A staining in the tumor nodules, suggesting that CMA is activated in HCC.

Figure 7

Shown is a schematic diagram illustrating how cellular adaptive response to HCV-induced ER stress results in degradation of the major tumor suppressors and autophagy inhibition. Autophagy inhibition activates oncogenic signaling at the membrane due to impaired EGFR degradation. The simultaneous loss of tumor suppressors p53, pRB, p21 and p14ARF results in cell growth and proliferation of HCC development in the cirrhotic livers. All these events promote HCC growth in liver cirrhosis.

Figure 8

Shown is a hypothetical model demonstrating how HCV-induced severe stress response impairs IFNAR1-p53-mediated innate antiviral loop that blocks transcription of type I IFN, ISGs, and IRFs. Low stress favors cellular p53-mediated cellular expression of ISGs and various IRFs that maintain the innate hepatic immunity and cellular apoptosis. Hepatic adaptive response to virus-associated stress selectively promotes CMA-associated degradation of IFNAR1, p53, and p14ARF and favors cell survival. Loss of IFNAR1 and p53 disables the activation of the innate feedback loop leading to severe impairment of innate immunity in the highly stressed cirrhotic liver.