Hai Huang

Elevated levels of mitochondrion-associated autophagy inhibitor LRPPRC are associated with poor prognosis in patients with prostate cancer

Autophagy has recently been found to play important roles in tumorigenesis and leucine‐rich pentatricopeptide repeat motif‐containing protein (LRPPRC) has been identified as an inhibitor that suppresses autophagy and mitophagy and maintains mitochondrial activity. The authors hypothesized that LRPPRC levels can be used as a biomarker for the diagnosis and prognosis of prostate cancer.

Matrine inhibits the proliferation, invasion and migration of castration-resistant prostate cancer cells through regulation of the NF-κB signaling pathway.

Matrine is a naturally occurring alkaloid extracted from the Chinese herb Sophora flavescens. It has been demonstrated to exhibit antiproliferative properties, promote apoptosis and inhibit cell invasion in a number of cancer cell lines. It has also been shown to improve the efficacy of chemotherapy when it is combined with other chemotherapy drugs. However, the therapeutic efficacy of matrine for prostate cancer remains poorly understood. In the present study, we showed that matrine inhibited the proliferation, migration and invasion of both DU145 and PC-3 cells in a dose- and time-dependent manner. It also reduced the cell population at S phase and increased the cell population at sub-G1 phase. The increases in both the apoptotic cell population and cell population at S and sub-G1 phases consistently indicated a pro-apoptotic effect of matrine. Decreases in levels of P65, p-P65, IKKα/β, p-IKKα/β, IKBα and p-IKBα as detected by immunoblot analysis in the matrine-treated DU145 and PC-3 cells suggested an involvement of the NF-κB signaling pathway. Therefore, it is a novel promising addition to the current arsenal of chemotherapy drugs for the treatment of androgen-independent prostate cancer.

Autophagy defects suggested by low levels of autophagy activator MAP1S and high levels of autophagy inhibitor LRPPRC predict poor prognosis of prostate cancer patients

MAP1S (originally named C19ORF5) is a widely distributed homolog of neuronal‐specific MAP1A and MAP1B, and bridges autophagic components with microtubules and mitochondria to affect autophagosomal biogenesis and degradation. Mitochondrion‐associated protein LRPPRC functions as an inhibitor for autophagy initiation to protect mitochondria from autophagy degradation. MAP1S and LRPPRC interact with each other and may collaboratively regulate autophagy although the underlying mechanism is yet unknown. Previously, we have reported that LRPPRC levels serve as a prognosis marker of patients with prostate adenocarcinomas (PCA), and that patients with high LRPPRC levels survive a shorter period after surgery than those with low levels of LRPPRC. MAP1S levels are elevated in diethylnitrosamine‐induced hepatocelular carcinomas in wildtype mice and the exposed MAP1S‐deficient mice develop more malignant hepatocellular carcinomas. We performed immunochemical analysis to evaluate the co‐relationship among the levels of MAP1S, LRPPRC, P62, and γ‐H2AX. Samples were collected from wildtype and prostate‐specific PTEN‐deficient mice, 111 patients with PCA who had been followed up for 10 years and 38 patients with benign prostate hyperplasia enrolled in hospitals in Guangzhou, China. The levels of MAP1S were generally elevated so the MAP1S‐mediated autophagy was activated in PCA developed in either PTEN‐deficient mice or patients than their respective benign tumors. The MAP1S levels among patients with PCA vary dramatically, and patients with low MAP1S levels survive a shorter period than those with high MAP1S levels. Levels of MAP1S in collaboration with levels of LRPPRC can serve as markers for prognosis of prostate cancer patients.

Defects in MAP1S-mediated autophagy cause reduction in mouse lifespans especially when fibronectin is overexpressed.

Autophagy is a cellular process that executes the turnover of dysfunctional organelles and misfolded or abnormally aggregated proteins. Microtubule‐associated protein MAP1S interacts with autophagy marker LC3 and positively regulates autophagy flux. LC3 binds with fibronectinmRNA and facilitates its translation. The synthesized fibronectin protein is exported to cell surface to initiate the assembly of fibronectin extracellular matrix. Fibronectin is degraded in lysosomes after it is engulfed into cytosol via endocytosis. Here, we show that defects in MAP1S‐mediated autophagy trigger oxidative stress, sinusoidal dilation, and lifespan reduction. Overexpression of LC3 in wild‐type mice increases the levels of fibronectin and γ‐H2AX, a marker of DNA double‐strand breakage. LC3‐induced fibronectin is efficiently degraded in lysosomes to maintain a balance of fibronectin levels in wild‐type mice so that the mice live a normal term of lifespan. In the LC3 transgenic mice with MAP1S deleted, LC3 enhances the synthesis of fibronectin but the MAP1S depletion causes an impairment of the lysosomal degradation of fibronectin. The accumulation of fibronectin protein promotes liver fibrosis, induces an accumulation of cell population at the G0/G1 stage, and further intensifies oxidative stress and sinusoidal dilatation. The LC3‐induced overexpression of fibronectin imposes stresses on MAP1S‐deficient mice and dramatically reduces their lifespans. Therefore, MAP1S‐mediated autophagy plays an important role in maintaining mouse lifespan especially in the presence of extra amount of fibronectin.

Elevation of SHARPIN Protein Levels in Prostate Adenocarcinomas Promotes Metastasis and Impairs Patient Survivals.

SHARPIN, SHANK‐associated RH domain interacting protein, associates with a linear ubiquitin chain assembly complex (LUBAC) to regulate inflammation and immunity. It has been reported that SHARPIN is highly expressed in several human tumors including ovarian cancer and liver cancer. We found that SHARPIN is also highly expressed in prostate cancer cell lines of DU145, LNCAP, and PC‐3. Suppression of SHARPIN caused an inhibition of NF‐κB signal and decreases in tumorigenesis of cultured cells in NOD/SCID mouse model. Overexpression of SHARPIN in prostate cancer cells promoted cell growth and reduced apoptosis through NF‐kB/ERK/Akt pathway and apoptosis‐associated proteins.

Elevated levels of epithelial cell transforming sequence 2 predicts poor prognosis for prostate cancer.

Epithelial cell transforming sequence 2 (Ect2) was originally reported as an oncogene that is involved in several types of human cancers. However, little is known about its expression and function in prostate cancer. Immunohistochemical staining for Ect2 was performed on a human tissue microarray. The staining intensity was analyzed in association with clinical pathological parameters such as Gleason score, pathological grade, clinical stage, tumor invasion, lymph node and distant metastasis. Furthermore, we repeated such analysis and investigated the prognostic value of Ect2 using the TCGA (The Cancer Genome Atlas) Dataset. Our immunohistochemical results showed that the expression levels of Ect2 protein were enhanced in human prostate cancer tissues. There existed positive correlations between the expression levels of Ect2 and several clinicopathological parameters, including advanced clinical stage, enhanced tumor invasion and lymph node metastasis. Similarly, we found that the expression levels of Ect2 were positively related to Gleason score, tumor invasion, lymph node metastasis and high distant metastasis in the TCGA Dataset. Kaplan–Meier analysis revealed that lower levels of Ect2 mRNA predicted higher overall survivals and biochemical recurrence (BCR)-free survivals in all patients or non-metastatic patients. Multivariate analysis by Cox regression showed that the expression of Ect2 could be an independent prognostic marker of poor BCR-free survivals. Therefore, levels of Ect2 may serve as a novel marker for the diagnosis or prognosis of prostate cancer.

Matrine suppresses invasion of castration-resistant prostate cancer cells by downregulating MMP-2/9 via NF-κB signaling pathway

Matrine is an alkaloid from Sophora flavescens that exhibits multiple protective effects on cancers. However, the molecular mechanisms of anti-metastatic effects of matrine on castration-resistant prostate cancer (CRPC) remain unknown. This study investigated the anti-metastatic effects of matrine on CRPC to identify the underlying mechanisms. The effects of matrine on the cell viability of DU145 and PC-3 cells were measured using MTS assay. The impact of matrine on expression levels of matrix metalloproteinase (MMP)-9, MMP-2, nuclear factor-κB (NF-κB) subunit p65 and phosphorylated p65 in cells untreated or treated with matrine were analyzed by western blotting. The inhibitory effects of matrine on cell migration and invasion were examined by Transwell assay. The impact of matrine on tumorigenesis in male Balb/c nude mice inoculated subcutaneously with cells were investigated in vivo. We found that matrine inhibited the growth of DU145 and PC3 cells time- and dose-dependently both inxa0vitro and in vivo. Migration and invasion capabilities of cells were also suppressed by matrine. At the same time, matrine markedly reduced the expression levels of MMP-9, MMP-2 and p-p65 in both cell lines. Further experiments revealed that matrine exhibited inhibitory effects of migration and invasion of CRPC by downregulating MMP-2/9 through NF-κB pathway. Matrine inhibits invasion of CRPC by reducing levels of MMP-9 and MMP-2 through NF-κB pathway. Therefore, it may be a potential anti-metastatic therapeutic agent for CRPC.

Defects in MAP1S-mediated autophagy turnover of fibronectin cause renal fibrosis

Excessive deposition of extracellular matrix proteins in renal tissues causes renal fibrosis and renal function failure. Mammalian cells primarily use the autophagy-lysosome system to degrade misfolded/aggregated proteins and dysfunctional organelles. MAP1S is an autophagy activator and promotes the biogenesis and degradation of autophagosomes. Previously, we reported that MAP1S suppresses hepatocellular carcinogenesis in a mouse model and predicts a better prognosis in patients suffering from clear cell renal cell carcinomas. Furthermore, we have characterized that MAP1S enhances the turnover of fibronectin, and mice overexpressing LC3 but with MAP1S deleted accumulate fibronectin and develop liver fibrosis because of the synergistic impact of LC3-induced over-synthesis of fibronectin and MAP1S depletion-caused impairment of fibronectin degradation. Here we show that a suppression of MAP1S in renal cells caused an impairment of autophagy clearance of fibronectin and an activation of pyroptosis. Depletion of MAP1S in mice leads to an accumulation of fibrosis-related proteins and the development of renal fibrosis in aged mice. The levels of MAP1S were dramatically reduced and levels of fibronectin were greatly elevated in renal fibrotic tissues from patients diagnosed as renal atrophy and renal failure. Therefore, MAP1S deficiency may cause the accumulation of fibronectin and the development of renal fibrosis.

Potassium Bisperoxo(1,10-phenanthroline)oxovanadate (bpV(phen)) Induces Apoptosis and Pyroptosis and Disrupts the P62-HDAC6 Protein Interaction to Suppress the Acetylated Microtubule-dependent Degradation of Autophagosomes.

Background: BpV(phen) is an insulin-mimetic small molecule. Results: We demonstrate that bpV(phen) reduces the stability of p62 in a proteasome-dependent way to activate HDAC6 to inhibit autophagy and induce apoptosis and pyroptosis. Conclusion: We propose that bpV(phen) inhibits autophagy through p62. Significance: We provide insights into a novel function of bpV(phen) and p62 in autophagy. Autophagy is a cellular process that controls and executes the turnover of dysfunctional organelles and misfolded or abnormally aggregated proteins. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activates the initiation of autophagy. Autophagosomes migrate along acetylated microtubules to fuse with lysosomes to execute the degradation of the engulfed substrates that usually bind with sequestosome 1 (SQSTM1, p62). Microtubule-associated protein 1 light chain 3 (LC3) traces the autophagy process by converting from the LC3-I to the LC3-II isoform and serves as a major marker of autophagy flux. Potassium bisperoxo(1,10-phenanthroline)oxovanadate (bpV(phen)) is an insulin mimic and a PTEN inhibitor and has the potential to treat different diseases. Here we show that bpV(phen) enhances the ubiquitination of p62, reduces the stability of p62, disrupts the interaction between p62 and histone deacetylase 6 (HDAC6), activates the deacetylase activity of HDAC6 on α-tubulin, and impairs stable acetylated microtubules. Microtubular destabilization leads to the blockade of autophagosome-lysosome fusion and accumulation of autophagosomes. Autophagy defects lead to oxidative stress and lysosomal rupture, which trigger different types of cell death, including apoptosis and pyroptosis. The consistent results from multiple systems, including mouse and different types of mammalian cells, are different from the predicted function of bpV(phen) as a PTEN inhibitor to activate autophagy flux. In addition, levels of p62 are reduced but not elevated when autophagosomal degradation is blocked, revealing a novel function of p62 in autophagy regulation. Therefore, it is necessary to pay attention to the roles of bpV(phen) in autophagy, apoptosis, and pyroptosis when it is developed as a drug.

The specific killing effect of matrine on castration-resistant prostate cancer cells by targeting the Akt/FoxO3a signaling pathway

Matrine, a Sophora alkaloid, exhibits antiproliferative and anti-carcinogenic activities through several mechanisms. In a previous study, we found that matrine could effectively inhibit the proliferation of castration-resistant prostate cancer (CRPC). In the present study, the effect of matrine and LY294002 on the expression of the Akt/FoxO3a signaling pathway was examined by western blot analyses and RT-PCR. We discovered that matrine significantly inhibited the proliferation of both prostate cancer cell line PC-3 and prostate epithelial cell line RWPE1, induced apoptosis and induced cell cycle arrest. In addition, LY294002 was found to enhance the effect of matrine. Furthermore, the effects of matrine on the inhibition of proliferation and the induction of cell cycle arrest and cell apoptosis were more effective on PC-3 than on RWPE1 cells. Compared to RWPE1 cells, matrine exerted a more powerful influence on PC-3 cells in increasing the expression of the relevant protein. Our data suggested that FoxO3a-Bim and FoxO3a-P27 may mediate matrine-inhibited proliferation of CRPC cells by activating cell apoptosis and inducing cell cycle arrest. Matrine exhibited high selectivity in killing CRPC cells. Our findings demonstrated that matrine could be used in a potential therapeutic role in the management of CRPC in humans.