GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation.

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5'-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

Wnt Signaling Stimulates Cooperation between GREB1 and HNF4α to Promote Proliferation in Hepatocellular Carcinoma.

Wnt signaling is known to maintain two cell states, hepatocyte differentiation and proliferation, in hepatocellular carcinoma (HCC). On the other hand, activation of Wnt signaling in colon cancer promotes uncontrollable stereotypic proliferation, whereas cells remain undifferentiated. To elucidate the unique mode of Wnt signaling in HCC, we comprehensively investigated HCC-specific Wnt pathway target genes and identified GREB1. Wnt signaling induced expression of GREB1 coupled with HNF4α and FOXA2, master transcription factors that maintain hepatic differentiation. Moreover, GREB1 was enriched at the regulatory region of atypical HNF4α target genes, including progrowth genes, thereby stimulating HCC proliferation. Therefore, GREB1 acts as a unique mediator of versatile Wnt signaling in HCC progression, bridging the roles of the Wnt pathway in differentiation and proliferation. SIGNIFICANCE: GREB1 is a liver cancer-specific Wnt signaling target gene that induces an oncogenic shift of HNF4α, a putative tumor suppressor, and may represent a therapeutic target in Wnt-activated hepatocellular carcinoma.

Localization of KRAS downstream target ARL4C to invasive pseudopods accelerates pancreatic cancer cell invasion.

Pancreatic cancer has a high mortality rate due to metastasis. Whereas KRAS is mutated in most pancreatic cancer patients, controlling KRAS or its downstream effectors has not been succeeded clinically. ARL4C is a small G protein whose expression is induced by the Wnt and EGF-RAS pathways. In the present study, we found that ARL4C is frequently overexpressed in pancreatic cancer patients and showed that its localization to invasive pseudopods is required for cancer cell invasion. IQGAP1 was identified as a novel interacting protein for ARL4C. ARL4C recruited IQGAP1 and its downstream effector, MMP14, to invasive pseudopods. Specific localization of ARL4C, IQGAP1, and MMP14 was the active site of invasion, which induced degradation of the extracellular matrix. Moreover, subcutaneously injected antisense oligonucleotide against ARL4C into tumor-bearing mice suppressed metastasis of pancreatic cancer. These results suggest that ARL4C-IQGAP1-MMP14 signaling is activated at invasive pseudopods of pancreatic cancer cells.

Most cases of pancreatic cancer are detected in the later stages when they are difficult to treat and, as a result, survival is low. Over 90% of pancreatic cancers contain genetic changes that increase the activity of a protein called KRAS. This hyperactive KRAS drives cancer growth and progression. Attempts to treat pancreatic cancer using drugs that reduce the activity of KRAS have so far failed. The KRAS protein can accelerate growth in healthy cells as well as in cancer and it does this by activating various other proteins. Drugs that target some of these other proteins could be more effective at treating pancreatic cancer than the drugs that target KRAS. One of these potential targets is called ARL4C. ARL4C is active during fetal development, but it is often not present in adult tissues. Harada et al. investigated whether the protein is important in pancreatic cancer, and what other roles it has in the body, to better understand if it is a good target for cancer treatment. First, Harada et al. used cells grown in the lab to show that ARL4C contributes to the aggressive spread of human pancreatic cancers. Using mice, Harada et al. also showed that blocking the activity of ARL4C in pancreatic cancers helped to slow their progression. Harada et al.'s results suggest that ARL4C could be a good target for new drugs treating pancreatic cancers. Given that this protein does not seem to have important roles in the cells of adults, targeting it is unlikely to have major side effects. Further investigation of ARL4C in more human-like animal models will help to confirm these results.

The Dickkopf1 and FOXM1 positive feedback loop promotes tumor growth in pancreatic and esophageal cancers.

Dickkopf1 (DKK1) is overexpressed in various cancers and promotes cancer cell proliferation by binding to cytoskeleton-associated protein 4 (CKAP4). However, the mechanisms underlying DKK1 expression are poorly understood. RNA sequence analysis revealed that expression of the transcription factor forkhead box M1 (FOXM1) and its target genes concordantly fluctuated with expression of DKK1 in pancreatic ductal adenocarcinoma (PDAC) cells. DKK1 knockdown decreased FOXM1 expression and vice versa in PDAC and esophageal squamous cell carcinoma (ESCC) cells. Inhibition of either the DKK1-CKAP4-AKT pathway or the ERK pathway suppressed FOXM1 expression, and simultaneous inhibition of both pathways showed synergistic effects. A FOXM1 binding site was identified in the 5'-untranslated region of the DKK1 gene, and its depletion decreased DKK1 expression and cancer cell proliferation. Clinicopathological and database analysis revealed that PDAC and ESCC patients who simultaneously express DKK1 and FOXM1 have a poorer prognosis. Multivariate analysis demonstrated that expression of both DKK1 and FOXM1 is the independent prognostic factor in ESCC patients. Although it has been reported that FOXM1 enhances Wnt signaling, FOXM1 induced DKK1 expression independently of Wnt signaling in PDAC and ESCC cells. These results suggest that DKK1 and FOXM1 create a positive feedback loop to promote cancer cell proliferation.