RSK2-inactivating mutations potentiate MAPK signaling and support cholesterol metabolism in hepatocellular carcinoma.

BACKGROUND & AIMS: Mutational profiling of patient tumors has suggested that hepatocellular carcinoma (HCC) development is mainly driven by loss-of-function mutations in tumor suppressor genes. p90 ribosomal S6 kinase 2 (RSK2) functions as a direct downstream kinase of ERK1/2 and elevated RSK2 expression has been reported to support oncogenic functions in some cancers. We investigated if RSK2 was also dysregulated by inactivating mutations in cancers including HCC. METHODS: We performed exome sequencing and targeted DNA sequencing on HBV-associated HCCs to examine recurrent RSK2 mutations. The functional significance and mechanistic consequences of RSK2 mutations were examined in natural RSK2-null HCC cells, and RSK2-knockout HCC cells. The potential downstream pathways underlying RSK2 mutations were investigated by RNA sequencing, qRT-PCR and mass spectrometry. RESULTS: We detected recurrent somatic RSK2 mutations at a rate of 6.3% in our HCC cohorts and revealed that, among many cancer types, HCC was the cancer most commonly harboring RSK2 mutations. The RSK2 mutations were inactivating and associated with a more aggressive tumor phenotype. We found that, functionally, restoring RSK2 expression in natural RSK2-null HBV-positive Hep3B cells suppressed proliferation and migration in vitro and tumorigenicity in vivo. Mechanistically, RSK2-inactivating mutations attenuated a SOS1/2-dependent negative feedback loop, leading to the activation of MAPK signaling. Of note, this RSK2 mutation-mediated MAPK upregulation rendered HCC cells more sensitive to sorafenib, a first-line multi-kinase inhibitor for advanced HCC. Furthermore, such activation of MAPK signaling enhanced cholesterol biosynthesis-related gene expression in HCC cells. CONCLUSIONS: Our findings reveal the mechanistic and functional significance of RSK2-inactivating mutations in HCC. These inactivating mutations may serve as an alternative route to activate MAPK signaling and cholesterol metabolism in HCC. LAY SUMMARY: In this study, we identified and functionally characterized RSK2-inactivating mutations in human hepatocellular carcinoma and demonstrated their association with aggressive tumor behavior. Mutations in RSK2 drive signaling pathways with known oncogenic potential, leading to enhanced cholesterol biosynthesis and potentially sensitizing tumors to sorafenib treatment.

Sorted-Cell Sequencing on HCC Specimens Reveals EPS8L3 as a Key Player in CD24/CD13/EpCAM-Triple Positive, Stemness-Related HCC Cells.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with varying levels of liver tumor initiating or cancer stem cells in the tumors. We aimed to investigate the expression of different liver cancer stem cell (LCSC) markers in human HCCs and identify their regulatory mechanisms in stemness-related cells. METHODS: We used an unbiased, single-marker sorting approach by flow cytometry, fluorescence-activated cell sorting, and transcriptomic analyses on HCC patients' resected specimens. Knockdown approach was used, and relevant functional assays were conducted on the identified targets of interest. RESULTS: Flow cytometry on a total of 60 HCC resected specimens showed significant heterogeneity in the expression of LCSC markers, with CD24, CD13, and EpCAM mainly contributing to this heterogeneity. Concomitant expression of CD24, CD13, and EpCAM was detected in 32 HCC samples, and this was associated with advanced tumor stages. Transcriptomic sequencing on the HCC cells sorted for these individual markers identified epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3) as a common gene associated with the 3 markers and was functionally validated in HCC cells. Knocking down EPS8L3 suppressed the expression of all 3 markers. To search for the upstream regulation of EPS8L3, we found SP1 bound to EPS8L3 promoter to drive EPS8L3 expression. Furthermore, using Akt inhibitor MK2206, we showed that Akt signaling-driven SP1 drove the expression of the 3 LCSC markers. CONCLUSIONS: Our findings suggest that Akt signaling-driven SP1 promotes EPS8L3 expression, which is critical in maintaining the downstream expression of CD24, CD13, and EpCAM. The findings provide insight into potential LCSC-targeting therapeutic strategies.

Reciprocal interactions between malignant cells and macrophages enhance cancer stemness and M2 polarization in HBV-associated hepatocellular carcinoma.

Background: The tumor microenvironment of cancers has emerged as a crucial component in regulating cancer stemness and plays a pivotal role in cell-cell communication. However, the specific mechanisms underlying these phenomena remain poorly understood. Methods: We performed the single-cell RNA sequencing (scRNA-seq) on nine HBV-associated hepatocellular carcinoma (HCC) patients. The heterogeneity of the malignant cells in pathway functions, transcription factors (TFs) regulation, overall survival, stemness, as well as ligand-receptor-based intercellular communication with macrophages were characterized. The aggressive and stemness feature for the target tumor subclone was validated by the conduction of in vitro assays including sphere formation, proliferation, Annexin V apoptosis, flow cytometry, siRNA library screening assays, and multiple in vivo preclinical mouse models including mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection. Results: Our analysis yielded a comprehensive atlas of 31,664 cells, revealing a diverse array of malignant cell subpopulations. Notably, we identified a stemness-related subclone of HCC cells with concurrent upregulation of CD24, CD47, and ICAM1 expression that correlated with poorer overall survival. Functional characterization both in vitro and in vivo validated S100A11 as one of the top downstream mediators for tumor initiation and stemness maintenance of this subclone. Further investigation of cell-cell communication within the tumor microenvironment revealed a propensity for bi-directional crosstalk between this stemness-related subclone and tumor-associated macrophages (TAMs). Co-culture study showed that this interaction resulted in the maintenance of the expression of cancer stem cell markers and driving M2-like TAM polarization towards a pro-tumorigenic niche. We also consolidated an inverse relationship between the proportions of TAMs and tumor-infiltrating T cells. Conclusions: Our study highlighted the critical role of stemness-related cancer cell populations in driving an immunosuppressive tumor microenvironment and identified the S100A11 gene as a key mediator for stemness maintenance in HCC. Moreover, our study provides support that the maintenance of cancer stemness is more attributed to M2 polarization than the recruitment of the TAMs.

Single-cell RNA sequencing shows the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma.

Interaction between tumor cells and immune cells in the tumor microenvironment is important in cancer development. Immune cells interact with the tumor cells to shape this process. Here, we use single-cell RNA sequencing analysis to delineate the immune landscape and tumor heterogeneity in a cohort of patients with HBV-associated human hepatocellular carcinoma (HCC). We found that tumor-associated macrophages suppress tumor T cell infiltration and TIGIT-NECTIN2 interaction regulates the immunosuppressive environment. The cell state transition of immune cells towards a more immunosuppressive and exhaustive status exemplifies the overall cancer-promoting immunocellular landscape. Furthermore, the heterogeneity of global molecular profiles reveals co-existence of intra-tumoral and inter-tumoral heterogeneity, but is more apparent in the latter. This analysis of the immunosuppressive landscape and intercellular interactions provides mechanistic information for the design of efficacious immune-oncology treatments in hepatocellular carcinoma.