Chromosome 8p engineering reveals increased metastatic potential targetable by patient-specific synthetic lethality in liver cancer.

Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33-mega-base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.

Prohibitin, STAT3 and SH2D4A physically and functionally interact in tumor cell mitochondria.

Chromosome 8p is frequently deleted in various cancer entities and has been shown to correlate with poor patient survival. SH2D4A is located on chromosome 8p and prevents the nuclear translocation of the pro-tumorigenic transcription factor STAT3. Here, we investigated the interaction of SH2D4A and STAT3 to shed light on the non-canonical functions of STAT3 in cooperation with the tumor suppressor SH2D4A. Using an immunoprecipitation-mass spectrometry (IP-MS) approach, we identified the mitochondrial scaffold proteins prohibitin 1 (PHB1) and prohibitin 2 (PHB2) among other proteins to potentially bind to SH2D4A. Co-immunoprecipitation and proximity ligation assays confirmed direct interactions of STAT3, PHB1, and SH2D4A in situ and in vitro. In addition, cell fractionation and immunofluorescence staining revealed co-localization of these proteins with mitochondria. These interactions were selectively interrupted by the small molecule and PHB ligand FL3. Furthermore, FL3 led to a reduction of STAT3 protein levels, STAT3 transcriptional activity, and HIF1α protein stabilization upon dimethyloxalylglycine (DMOG) treatment. Besides, mitochondrial fusion and fission markers, L-OPA1, Mfn1, and FIS1, were dysregulated upon FL3 treatment. This dysregulated morphology was accompanied by significant reduction of mitochondrial respiration, thus, FL3 significantly diminished mitochondrial respirational capacity. In contrast, SH2D4A knockout increased mitochondrial respiration, whereas FL3 reversed the effect of SH2D4A knockout. The here described results indicate that the interaction of SH2D4A and PHB1 is involved in the mitochondrial function and integrity. The demonstrated interaction with STAT3, accompanied by its reduction of transcriptional activity, further suggests that SH2D4A is linking STAT3 to its mitochondrial functions, and inhibition of PHB-interaction may have therapeutic effects in tumor cells with STAT3 activation.

NOTCH target gene HES5 mediates oncogenic and tumor suppressive functions in hepatocarcinogenesis.

NOTCH receptor signaling plays a pivotal role in liver homeostasis and hepatocarcinogenesis. However, the role of NOTCH pathway mutations and the NOTCH target gene HES5 in liver tumorigenesis are poorly understood. Here we performed whole-exome sequencing of 54 human HCC specimens and compared the prevalence of NOTCH pathway component mutations with the TCGA-LIHC cohort (N = 364). In addition, we functionally characterized the NOTCH target HES5 and the patient-derived HES5-R31G mutation in vitro and in an orthotopic mouse model applying different oncogenic backgrounds, to dissect the role of HES5 in different tumor subgroups in vivo. We identified nonsynonymous mutations in 14 immediate NOTCH pathway genes affecting 24.1% and 16.8% of HCC patients in the two independent cohorts, respectively. Among these, the HES5-R31G mutation was predicted in silico to have high biological relevance. Functional analyses in cell culture showed that HES5 reduced cell migration and clonogenicity. Further analyses revealed that the patient-derived HES5-R31G mutant protein was non-functional due to loss of DNA binding and greatly reduced nuclear localization. Furthermore, HES5 exhibited a negative feedback loop by directly inhibiting the NOTCH target HES1 and downregulated the pro-proliferative MYC targets ODC1 and LDHA. Interestingly, HES5 inhibited MYC-dependent hepatocarcinogenesis, whereas it promoted AKT-dependent liver tumor formation and stem cell features in a murine model. Thus, NOTCH pathway component mutations are commonly observed in HCC. Furthermore, the NOTCH target gene HES5 has both pro- and anti-tumorigenic functions in liver cancer proposing a driver gene dependency and it promotes tumorigenesis with its interaction partner AKT.