Genetic assessment of pathogenic germline alterations in lysosomal genes among Asian patients with pancreatic ductal adenocarcinoma.

BACKGROUND: Lysosomes are closely linked to autophagic activity, which plays a vital role in pancreatic ductal adenocarcinoma (PDAC) biology. The survival of PDAC patients is still poor, and the identification of novel genetic factors for prognosis and treatment is highly required to prevent PDAC-related deaths. This study investigated the germline variants related to lysosomal dysfunction in patients with PDAC and to analyze whether they contribute to the development of PDAC. METHODS: The germline putative pathogenic variants (PPV) in genes involved in lysosomal storage disease (LSD) was compared between patients with PDAC (n = 418) and healthy controls (n = 845) using targeted panel and whole-exome sequencing. Furthermore, pancreatic organoids from wild-type and KrasG12D mice were used to evaluate the effect of lysosomal dysfunction on PDAC development. RNA sequencing (RNA-seq) analysis was performed with established PDAC patient-derived organoids (PDOs) according to the PPV status. RESULTS: The PPV in LSD-related genes was higher in patients with PDAC than in healthy controls (8.13 vs. 4.26%, Log2 OR = 1.65, P = 3.08 × 10-3). The PPV carriers of LSD-related genes with PDAC were significantly younger than the non-carriers (mean age 61.5 vs. 65.3 years, P = 0.031). We further studied a variant of the lysosomal enzyme, galactosylceramidase (GALC), which was the most frequently detected LSD variant in our cohort. Autophagolysosomal activity was hampered when GALC was downregulated, which was accompanied by paradoxically elevated autophagic flux. Furthermore, the number of proliferating Ki-67+ cells increased significantly in pancreatic organoids derived from Galc knockout KrasG12D mice. Moreover, GALC PPV carriers tended to show drug resistance in both PDAC cell line and PDAC PDO, and RNA-seq analysis revealed that various metabolism and gene repair pathways were upregulated in PDAC PDOs harboring a GALC variant. CONCLUSIONS: Genetically defined lysosomal dysfunction is frequently observed in patients with young-onset PDAC. This might contribute to PDAC development by altering metabolism and impairing autophagolysosomal activity, which could be potentially implicated in therapeutic applications for PDAC.

CDK9 inhibitors downregulate DKK1 expression to suppress the metastatic potential of HCC cells.

BACKGROUND: Elevated expression of Dickkopf-1 (DKK1) is frequently observed in hepatocellular carcinoma (HCC) patients with poor clinical outcomes. Several reports indicating the functional involvement of DKK1 in HCC progression have suggested DKK1 as a promising therapeutic target for HCC. OBJECTIVE: In this study, to develop an efficient way to target DKK1, we assessed the effect of CDK9 inhibitors on DKK1 expression linked to metastatic movement of HCC. METHODS: The expression of DKK1 in CDK9 inhibitor-treated HCC cells was measured by western blot, ELISA and quantitative real-time reverse transcription PCR. Wound healing assay, migration assay, invasion assay and western blot were examined to evaluate the functional role of DKK1 in CDK9 inhibitors-treated HCC. RESULTS: Inactivation of CDK9 either by a catalytic inhibitor being clinically evaluated or by a specific CDK9 protein degrader largely downregulated DKK1 expression at the transcript and protein levels. In addition, CDK9 inhibitors suppressed the migration and invasion of HCC cells. We observed that ectopic high expression of DKK1 at least partially reversed the defects in metastatic movement of HCC cells mediated by CDK9 inhibitors. We further discovered that the DKK1-nuclear ß-catenin axis associated with the metastatic potential of HCC cells was impaired by CDK9 inhibitors. CONCLUSION: Taken together, our findings suggest that CDK9 inhibitors are potent tools to target DKK1, which can suppress the metastatic progression of HCC.

Downregulation of SETD5 Suppresses the Tumorigenicity of Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is an aggressive and incurable cancer. Although understanding of the molecular pathogenesis of HCC has greatly advanced, therapeutic options for the disease remain limited. In this study, we demonstrated that SETD5 expression is positively associated with poor prognosis of HCC and that SETD5 depletion decreased HCC cell proliferation and invasion while inducing cell death. Transcriptome analysis revealed that SETD5 loss downregulated the interferon-mediated inflammatory response in HCC cells. In addition, SETD5 depletion downregulated the expression of a critical glycolysis gene, PKM (pyruvate kinase M1/2), and decreased glycolysis activity in HCC cells. Finally, SETD5 knockdown inhibited tumor growth in xenograft mouse models. These results collectively suggest that SETD5 is involved in the tumorigenic features of HCC cells and that targeting SETD5 may suppress HCC progression.

Synergistic antitumor activity of sorafenib and MG149 in hepatocellular carcinoma cells.

Advanced hepatocellular carcinoma (HCC) is among the most challenging cancers to overcome, and there is a need for better therapeutic strategies. Among the different cancer drugs that have been used in clinics, sorafenib is considered the standard first-line drug for advanced HCC. Here, to identify a chemical compound displaying a synergistic effect with sorafenib in HCC, we screened a focused chemical library and found that MG149, a histone acetyltransferase inhibitor targeting the MYST family, exhibited the most synergistic anticancer effect with sorafenib on HCC cells. The combination of sorafenib and MG149 exerted a synergistic anti-proliferation effect on HCC cells by inducing apoptotic cell death. We revealed that cotreatment with sorafenib and MG149 aggravated endoplasmic reticulum (ER) stress to promote the death of HCC cells rather than adaptive cell survival. In addition, combined treatment with sorafenib and MG149 significantly increased the intracellular levels of unfolded proteins and reactive oxygen species, which upregulated ER stress. Collectively, these results suggest that MG149 has the potential to improve the efficacy of sorafenib in advanced HCC via the upregulation of cytotoxic ER stress. [BMB Reports 2022; 55(10): 506-511].

LAD1 expression is associated with the metastatic potential of colorectal cancer cells.

BACKGROUND: Anchoring filament protein ladinin-1 (LAD1) was related to the aggressive progression of breast, lung, laryngeal and thyroid cancers. However, the association of LAD1 with colorectal cancer remained unknown. Here, to determine the relationship of LAD1 with colorectal cancer progression, we explored the effect of LAD1 loss on the malignant features of colorectal cancer cells. METHODS: We constructed LAD1-depleted cell lines and examined the effect of LAD1 deficiency on the phenotypic and molecular features of colorectal cancer cells in vitro. The function of LAD1 in metastasis in vivo was examined by establishing a spleen-to-liver metastasis mouse model. LAD1 protein expression in colorectal cancer patient specimens was assessed by immunohistochemistry of tumor microarrays. RESULTS: We found that LAD1 was abundant in most colorectal cancer cells. In addition, high expression of LAD1 significantly correlated with poor patient outcome. LAD1 depletion inhibited the migration and invasion of two different colorectal cancer cell lines, SW620 and Caco-2, without affecting their proliferation. In addition, LAD1 loss led to defects in liver metastasis of SW620 cells in the mouse model. Immunohistochemistry of colorectal cancer tissues revealed LAD1 enrichment in metastatic tissues compared to that in primary tumor and normal tissues. CONCLUSION: These results suggest that LAD1 expression is associated with the metastatic progression of colorectal cancer by promoting the migration and invasion of cancer cells.

KDM3A regulates Slug expression to promote the invasion of MCF7 breast cancer cells in hypoxia.

Oxygen deprivation (hypoxia), which frequently occurs in the tumour microenvironment, is a strong driver of the phenotypic transition of cancer cells. An increase in metastatic potential such as cell invasion is a well-known phenotypical change induced in hypoxia. The present study demonstrated that lysine demethylase 3A (KDM3A), a Jumonji C domain-containing KDM, is involved in the hypoxia-induced invasion of MCF7 breast cancer cells. KDM3A depletion inhibits the induction of cell invasion without affecting MCF7 cell survival rate or proliferation under hypoxic conditions, whereas KDM3A overexpression enhances MCF7 cell invasion even under normoxic conditions. KDM3A suppresses E-cadherin expression, which is associated with epithelial-to-mesenchymal transition (EMT)-mediated cell invasion in hypoxia. In addition, KDM3A promotes the expression of Slug, an EMT transcription factor that negatively regulates E-cadherin expression. Consistent with this finding, the removal of the repressive transcription marker, dimethylated histone H3 at lysine 9 from the Slug promoter is dependent on hypoxia-induced recruitment of KDM3A. Collectively, the results of the present study suggest that KDM3A is a crucial transcriptional coactivator of Slug expression to induce MCF7 breast cancer cell invasion in hypoxia, and that inhibition of KDM3A may efficaciously prevent metastatic cancer development.

Regulation of hypoxia responses by flavin adenine dinucleotide-dependent modulation of HIF-1α protein stability.

Oxygen deprivation induces a range of cellular adaptive responses that enable to drive cancer progression. Here, we report that lysine-specific demethylase 1 (LSD1) upregulates hypoxia responses by demethylating RACK1 protein, a component of hypoxia-inducible factor (HIF) ubiquitination machinery, and consequently suppressing the oxygen-independent degradation of HIF-1α. This ability of LSD1 is attenuated during prolonged hypoxia, with a decrease in the cellular level of flavin adenine dinucleotide (FAD), a metabolic cofactor of LSD1, causing HIF-1α downregulation in later stages of hypoxia. Exogenously provided FAD restores HIF-1α stability, indicating a rate-limiting role for FAD in LSD1-mediated HIF-1α regulation. Transcriptomic analyses of patient tissues show that the HIF-1 signature is highly correlated with the expression of LSD1 target genes as well as the enzymes of FAD biosynthetic pathway in triple-negative breast cancers, reflecting the significance of FAD-dependent LSD1 activity in cancer progression. Together, our findings provide a new insight into HIF-mediated hypoxia response regulation by coupling the FAD dependence of LSD1 activity to the regulation of HIF-1α stability.

Twist1 and AP-1 cooperatively upregulate integrin α5 expression to induce invasion and the epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is an important process implicated in tumor invasion and metastasis. Twist1 is a transcription factor that induces EMT, including E-cadherin suppression and cancer cell migration and invasion; hence it promotes cancer metastasis. Twist1 directly or indirectly regulates the expression of various genes and cellular functions involved in cancer progression. However, the underlying mechanisms remain largely unknown. In this study, we investigated the molecular basis for Twist1-mediated invasion and EMT. In human cancer cells, Twist1 was found to directly upregulate transcription of the mesenchymal gene integrin α5 in an E-box-independent, but activating protein-1 (AP-1) element-dependent, manner. Twist1 activated the integrin α5 promoter by interacting with and activating the transcription factor AP-1, composed of c-Jun and activating transcription factor-2 (ATF-2); it also enhanced the nuclear presence of ATF-2. AP-1 was critical for Twist1-induced cancer cell invasion, primarily through the induction of integrin α5, which activated c-Jun N-terminal kinase and focal adhesion kinase-signaling activities. Using data from The Cancer Genome Atlas, we found that Twist1 expression positively correlates with integrin α5 expression in human colorectal cancers. These findings suggest that cooperation between Twist1 and AP-1 represents a novel mechanism for EMT and tumor invasiveness. This study supports further investigation into the molecular basis underlying the diverse Twist1-mediated functions that occur during tumor progression.