The role of liver cancer stem cells in hepatocellular carcinoma metastasis.

Metastasis accounts for the vast majority of cancer deaths; however, this complex process has yet to be fully explained. To form metastases, cancer cells must undergo a series of steps, known as the "Metastatic cascade", each of which requires a specific functional transformation. Cancer stem cells (CSCs) play a vital role in tumor metastasis, but their dynamic behavior and regulatory mechanisms have not been fully elucidated. Based on the "Metastatic cascade" theory, this review summarizes the effect of liver CSCs on the metastatic biological programs that underlie the dissemination and metastatic growth of cancer cells. Liver CSCs have the capacity to initiate distant organ metastasis via EMT, and the microenvironment transformation that supports the ability of these cells to disseminate, evade immune surveillance, dormancy, and regenerate metastasis. Understanding the heterogeneity and traits of liver CSCs in these processes is critical for developing strategies to prevent and treat metastasis of advanced hepatocellular carcinoma (HCC).

The role of CD133 in hepatocellular carcinoma.

Cancer stem cells (CSCs) represent a small subpopulation of cells found within tumors that exhibit properties of self-renewal, like normal stem cells. CSCs have been defined as a crucial factor involved in driving cancer relapse, chemoresistance and metastasis. Prominin-1 (CD133) is one of the most well-characterized markers of CSCs in various tumor types, including hepatocellular carcinoma (HCC). CD133+ cells have been demonstrated to be involved in metastasis, tumorigenesis, tumor recurrence, and resistance to treatment in HCC. CD133-related clinical prognosis prediction, and targeted therapy have highlighted the clinical significance of CD133 in HCC. However, there remains controversy over the role of CD133 in experimental and clinical research involving HCC. In this article, we summarize the fundamental cell biology of CD133 in HCC cells and discuss the important characteristics of CD133+ in HCC cells. Furthermore, the prognostic value of CD133, and therapeutic strategies for its targeting in HCC, is also reviewed.

PIK3C3 regulates the expansion of liver CSCs and PIK3C3 inhibition counteracts liver cancer stem cell activity induced by PI3K inhibitor.

The existence of cancer stem cells (CSCs) accounts for hepatocellular carcinoma (HCC) treatment resistance, relapse, and metastasis. Although the elimination of cancer stem cells is crucial for cancer treatment, strategies for their elimination are limited. Here, we report that a remarkable increase in PIK3C3 was detected in HCC tissues and liver CSCs. Upregulated PIK3C3 facilitated liver CSC expansion in HCC cells; RNA interference-mediated silencing of PIK3C3 had an opposite effect. Furthermore, PIK3C3 inhibition by inhibitors effectively eliminated liver CSCs and inhibited the growth of tumors in vivo. The phosphoinositide 3-kinase (PI3K) pathway is considered an important hallmark of cancer. One of our recent studies found that prolonged inhibition by inhibitors of class I PI3K induces liver CSCs expansion. To our surprise, PIK3C3 inhibition blocked the expansion of CSCs induced by PI3K inhibitor; moreover, treatment with the combination of PIK3C3 inhibitor and PI3K inhibitor in maximal suppresses the expansion of liver CSCs of tumors in mice. Mechanistically, inhibition of PIK3C3 inhibit the activation of SGK3, a CSCs promoter, induced by PI3K inhibitor. We also show that PIK3C3 inhibitor suppresses liver CSCs by activation of the AMP-activated kinase (AMPK). Although PIK3C3 plays a critical role in autophagy, we find that PIK3C3 regulates liver CSCs independent of the autophagy process. These findings uncover the effective suppression of liver CSCs by targeting PIK3C3, and targeting PIK3C3 in combination with PI3K inhibitor inhibits the expansion of liver CSCs efficiently, which is an attractive therapeutic regimen for the treatment of HCC.

Aquaporin-9, Mediated by IGF2, Suppresses Liver Cancer Stem Cell Properties via Augmenting ROS/ß-Catenin/FOXO3a Signaling.

Liver cancer stem cells (LCSCs) play a critical role in hepatocellular carcinoma (HCC) by virtue of their aggressive behavior and association with poor prognoses. Aquaporin-9 (AQP9) is a transmembrane protein that transports water and reportedly transports H2O2. Recent studies have shown that AQP9 expression has a negative effect on HCC cell invasion by inhibiting the epithelial-to-mesenchymal transition. However, the role of AQP9 in LCSCs remains obscure. We performed spheroid formation assay and flow cytometric analysis to investigate LCSCs stemness. CD133+ and CD133- cells were isolated by flow cytometry. Real-time quantitative PCR (qRT-PCR), Western blot analysis, and immunofluorescence assay were used to estimate gene expression. The protein association of ß-catenin with TCF4 and the interaction of ß-catenin with FOXO3a were detected by immunoprecipitation (IP). Here, we found that AQP9 was preferentially decreased in LCSCs. Upregulated AQP9 significantly suppressed LCSCs stemness. In contrast, the inhibition of AQP9 had the opposite effect. Mechanistically, AQP9 was shown to be downregulated by insulin-like growth factor 2 (IGF2), which was widely reported to contribute to maintaining CSCs stemness. Furthermore, AQP9 overexpression was found to result in reactive oxygen species (ROS) accumulation, which inhibited ß-catenin activity by attenuating the interaction of ß-catenin with TCF4 while concurrently enhancing the association of ß-catenin with FOXO3a, ultimately inhibiting LCSCs stemness. Our study implies that stimulation of the AQP9 signaling axis may be a novel preventive and/or therapeutic approach for eliminating LCSCs. IMPLICATIONS: Our findings demonstrate that AQP9 signaling axis may be a novel preventive and/or therapeutic approach for eliminating LCSCs.

AQP9 suppresses hepatocellular carcinoma cell invasion through inhibition of hypoxia-inducible factor 1α expression under hypoxia.

BACKGROUND AND AIM: Intratumor hypoxia is a hallmark of hepatocellular carcinoma (HCC) and is associated with an aggressive tumor phenotype. Although it has been shown that AQP9 plays an important role in HCC, the relevance between hypoxia and AQP9 is still unknown. METHODS: We established in vitro normoxic or hypoxic models to investigate the role of AQP9 in the regulation of hypoxia-inducible factor 1α (HIF-1α) and hypoxia-enhanced invasion of hepatoma cells. Molecular expression was detected using western blot or quantitative polymerase chain reaction. Cell invasion ability was determined using Transwell invasion assay. In vivo xenograft experiment was used to detect the role of AQP9 on tumor growth. RESULTS: Our present study revealed a decrease in the expression levels of AQP9 in hypoxic microenvironments. Overexpression of AQP9 led to a decreased expression of HIF-1α; conversely, suppression of AQP9 in HCC cells had an opposite effect. Furthermore, up-regulated AQP9 blocked the hypoxic-enhanced invasion of HCC cells. The overexpression of AQP9 inhibited the growth of tumors and HIF-1α expression in vivo. CONCLUSIONS: These data suggest that AQP9 acts as a tumor suppressor in HCC invasion via the regulation of HIF-1α expression in the tumor hypoxic microenvironment.

Prolonged inhibition of class I PI3K promotes liver cancer stem cell expansion by augmenting SGK3/GSK-3ß/ß-catenin signalling.

BACKGROUND: Serum and glucocorticoid-regulated kinase 3 (SGK3) has been reported to play an important role in tumour progression, but its role in cancer stem cells (CSCs) remains obscure. The phosphoinositide 3-kinase (PI3K) pathway is considered a hallmark of cancer. Although many PI3K pathway-targeted therapies have been tested in oncology trials, the results are not satisfactory. METHODS: We used spheroids cultured in serum-free culture medium and MicroBead isolation to obtain liver CSCs. Spheroid formation assay and flow cytometric analysis were performed to investigate liver CSC expansion. Real-time polymerase chain reaction (PCR), western blot and immunofluorescence were used to assess gene expression in cell lines. RESULTS: We found that SGK3 is preferentially activated in liver CSCs. Upregulated SGK3 significantly increases the expansion of liver CSCs. Conversely, suppression of SGK3 in human hepatocarcinoma (HCC) cells had an opposite effect. Mechanistically, SGK3 promoted ß-catenin accumulation by suppressing GSK-3ß-mediated ß-catenin degradation in liver CSCs, and then promoting the expansion of liver CSCs. Prolonged treatment of HCC cells with class I PI3K inhibitors leads to activation of SGK3 and expansion of liver CSCs. Inhibition of hVps34 can block SGK3 activity and suppress liver CSC expansion induced by PI3K inhibitors. More importantly, we also found that prolonged treatment of HCC cells with PI3K inhibitors stimulates the ß-catenin signalling pathway via activation of SGK3. CONCLUSIONS: Prolonged inhibition of class I PI3K promotes liver CSC expansion by augmenting SGK3-dependent ß-catenin stabilisation, and effective inhibition of SGK3 signalling may be useful in eliminating liver CSCs and in PI3K pathway-targeted cancer therapies.

Detection of urine metabolites in polycystic ovary syndrome by UPLC triple-TOF-MS.

BACKGROUND: Polycystic ovary syndrome (PCOS) is an endocrine disorder whose pathogenesis remains unclear. There are also no effective biomarkers for this disease. We evaluated the metabolic changes in PCOS patients and to investigate potential metabolic biomarkers for PCOS. METHODS: Twenty-two women with PCOS and 15 healthy controls were studied. Urine samples were assessed through ultra-performance liquid chromatography-mass spectrometry followed by principal component analysis and partial least squares discriminant analysis. RESULTS: Using the presented methods, 59 urine metabolites were found at different concentrations in PCOS patients. Moreover, two novel potential biomarkers, testosterone-glucuronide and 11α-hydroxyprogesterone, and four candidate biomarkers, benzofenap, methionyl-phenylalanine, MG(18:4(6Z,9Z,12Z,15Z)/0:0/0:0) and 2-(14,15-epoxyeicosatrienoyl) glycerol, were found to show significant differences through variance analysis (P<0.01) and were identified as target metabolites. The two potential biomarkers identified in this study highly correlate to the metabolites catalyzed by the ovarian cytochrome P450c17α. CONCLUSIONS: Two novel potential urinary biomarkers, testosterone-glucuronide and 11α-hydroxyprogesterone, and four candidate urinary biomarkers, benzofenap, methionyl-phenylalanine, MG(18:4(6Z,9Z,12Z,15Z)/0:0/0:0), and 2-(14,15-epoxyeicosatrienoyl) glycerol, were identified in PCOS patients by a metabolomic approach. Further study of the biomarkers using larger populations is needed to validate these biomarkers and thereby understand the pathogenesis of PCOS, potentially allowing for its diagnosis.