Current Status of Patient-Derived Ovarian Cancer Models

Ovarian cancer (OC) is one of the leading causes of female cancer death. Recent studies have documented its extensive variations as a disease entity, in terms of cell or tissue of origin, pre-cancerous lesions, common mutations, and therapeutic responses, leading to the notion that OC is a generic term referring to a whole range of different cancer subtypes. Despite such heterogeneity, OC treatment is stereotypic; aggressive surgery followed by conventional chemotherapy could result in chemo-resistant diseases. Whereas molecular-targeted therapies will become shortly available for a subset of OC, there still remain many patients without effective drugs, requiring development of groundbreaking therapeutic agents. In preclinical studies for drug discovery, cancer cell lines used to be the gold standard, but now this has declined due to frequent failure in predicting therapeutic responses in patients. In this regard, patient-derived cells and tumors are gaining more attention in precise and physiological modeling of in situ tumors, which could also pave the way to implementation of precision medicine. In this article, we comprehensively overviewed the current status of various platforms for patient-derived OC models. We highly appreciate the potentials of organoid culture in achieving high success rate and retaining tumor heterogeneity

The splicing-factor oncoprotein SF2/ASF activates mTORC1

The splicing factor SF2/ASF is an oncoprotein that is up-regulated in many cancers and can transform immortal rodent fibroblasts when slightly overexpressed. The mTOR signaling pathway is activated in many cancers, and pharmacological blockers of this pathway are in clinical trials as anticancer drugs. We examined the activity of the mTOR pathway in cells transformed by SF2/ASF and found that this splicing factor activates the mTORC1 branch of the pathway, as measured by S6K and eIF4EBP1 phosphorylation. This activation is specific to mTORC1 because no activation of Akt, an mTORC2 substrate, was detected. mTORC1 activation by SF2/ASF bypasses upstream PI3K/Akt signaling and is essential for SF2/ASF-mediated transformation, as inhibition of mTOR by rapamycin blocked transformation by SF2/ASF in vitro and in vivo. Moreover, shRNA-mediated knockdown of mTOR, or of the specific mTORC1 and mTORC2 components Raptor and Rictor, abolished the tumorigenic potential of cells overexpressing SF2/ASF. These results suggest that clinical tumors with SF2/ASF up-regulation could be especially sensitive to mTOR inhibitors

β-Catenin/TCF4 Complex-Mediated Induction of the NRF3 (NFE2L3) Gene in Cancer Cells

Remarkable upregulation of the NRF2 (NFE2L2)-related transcription factor NRF3 (NFE2L3) in several cancer tissues and its correlation with poor prognosis strongly suggest the physiological function of NRF3 in tumors. Indeed, we had recently uncovered the function of NRF3, which promotes cancer cell proliferation by p53 degradation via the 20S proteasome. Nevertheless, the molecular mechanism underlying the induction of NRF3 gene expression in cancer cells is highly elusive. We herein describe that NRF3 upregulation is induced by the β-catenin/TCF4 complex in colon cancer cells. We first confirmed high NRF3 mRNA expression in human colon cancer specimens. The genome database indicated that the human NRF3 gene possesses a species-conserved WRE sequence (TCF/LEF consensus element), implying that the β-catenin/TCF complex activates NRF3 expression in colon cancer. Consistently, we observed that the β-catenin/TCF4 complex mediates NRF3 expression by binding directly to the WRE site. Furthermore, inducing NRF3 activates cell proliferation and the expression of the glucose transporter GLUT1. The existence of the β-catenin/TCF4-NRF3 axis was also validated in the intestine and organoids of Apc-deficient mice. Finally, the positive correlation between NRF3 and β-catenin target gene expression strongly supports our conclusion. Our findings clearly demonstrate that NRF3 induction in cancer cells is controlled by the Wnt/β-catenin pathway

Hepatocyte growth factor activator inhibitor-2 stabilizes Epcam and maintains epithelial organization in the mouse intestine

Makiko Kawaguchi et al. developed an inducible Spint2 knockout mouse model which exhibited extensive damage to the intestinal epithelium and resulted in death six days after tamoxifen-induced gene ablation. The extreme phenotype observed in this inducible line suggests an important role for Spint2 in maintenance of healthy intestinal epithelium

Establishment and Molecular Phenotyping of Organoids from the Squamocolumnar Junction Region of the Uterine Cervix

The metaplastic epithelium of the transformation zone (TZ) including the squamocolumnar junction (SCJ) of the uterine cervix is a prime target of human papilloma virus (HPV) infection and subsequent cancer development. Due to the lack of adequate in vitro models for SCJ, however, investigations into its physiological roles and vulnerability to carcinogenesis have been limited. By using Matrigel-based three-dimensional culture techniques, we propagated organoids derived from the normal SCJ region, along with metaplastic squamous cells in the TZ. Consisting predominantly of squamous cells, organoids basically exhibited a dense structure. However, at least in some organoids, a small but discrete population of mucin-producing endocervix cells co-existed adjacent to the squamous cell population, virtually recapitulating the configuration of SCJ in a TZ background. In addition, transcriptome analysis confirmed a higher expression level of many SCJ marker genes in organoids, compared to that in the immortalized cervical cell lines of non-SCJ origin. Thus, the obtained organoids appear to mimic cervical SCJ cells and, in particular, metaplastic squamous cells from the TZ, likely providing a novel platform in which HPV-driven cervical cancer development could be investigated

Interleukin-1β Expression in Human Gastric Carcinoma with Epstein-Barr Virus Infection

The KT tumor is a transplantable strain of a human Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), established in severe combined immunodeficiency (SCID) mice, with which the cytokine expression of EBVaGC can be investigated without interference from the infiltrating lymphocytes. As a part of a high-density oligonucleotide array (GeneChip) analysis of EBVaGC, the interleukin-1β (IL-1β) gene was the only cytokine gene that showed markedly higher expression in the KT tumor cells than in two tumor strains of EBV-negative GC. The results were confirmed by Northern blotting, Western blotting, and enzyme-linked immunosorbent assay. Furthermore, we demonstrated a positive signal for IL-1β mRNA in the carcinoma cells of a surgically resected EBVaGC, but not in EBV-negative GC, by in situ hybridization. In vitro, IL-1β increased the cell growth of a GC cell line, TMK1. Thus, IL-1β may act as an autocrine growth factor in EBVaGC

Image_7_Inhibition of OCT4 binding at the MYCN locus induces neuroblastoma cell death accompanied by downregulation of transcripts with high-open reading frame dominance.tiff

Amplification of MYCN is observed in high-risk neuroblastomas (NBs) and is associated with a poor prognosis. MYCN expression is directly regulated by multiple transcription factors, including OCT4, MYCN, CTCF, and p53 in NB. Our previous study showed that inhibition of p53 binding at the MYCN locus induces NB cell death. However, it remains unclear whether inhibition of alternative transcription factor induces NB cell death. In this study, we revealed that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4–MYCN positive feedback loop, induces caspase-2-mediated cell death in MYCN-amplified NB. We used the CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in the MYCN-amplified NB cell lines CHP134 and IMR32. In both cell lines, the inhibition of OCT4 binding at the MYCN locus reduced MYCN expression, thereby suppressing MYCN-target genes. After inhibition of OCT4 binding, differentially downregulated transcripts were associated with high-open reading frame (ORF) dominance score, which is associated with the translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Furthermore, transcripts with a high-ORF dominance score were significantly associated with genes whose high expression is associated with a poor prognosis in NB. Because the ORF dominance score correlates with the translation efficiency of transcripts, our findings suggest that MYCN maintains the expression of transcripts with high translation efficiency, contributing to a poor prognosis in NB. In conclusion, the inhibition of OCT4 binding at the MYCN locus resulted in reduced MYCN activity, which in turn led to the downregulation of high-ORF dominance transcripts and subsequently induced caspase-2-mediated cell death in MYCN-amplified NB cells. Therefore, disruption of the OCT4 binding at the MYCN locus may serve as an effective therapeutic strategy for MYCN-amplified NB.</p

Image_2_Inhibition of OCT4 binding at the MYCN locus induces neuroblastoma cell death accompanied by downregulation of transcripts with high-open reading frame dominance.tiff

Amplification of MYCN is observed in high-risk neuroblastomas (NBs) and is associated with a poor prognosis. MYCN expression is directly regulated by multiple transcription factors, including OCT4, MYCN, CTCF, and p53 in NB. Our previous study showed that inhibition of p53 binding at the MYCN locus induces NB cell death. However, it remains unclear whether inhibition of alternative transcription factor induces NB cell death. In this study, we revealed that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4–MYCN positive feedback loop, induces caspase-2-mediated cell death in MYCN-amplified NB. We used the CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in the MYCN-amplified NB cell lines CHP134 and IMR32. In both cell lines, the inhibition of OCT4 binding at the MYCN locus reduced MYCN expression, thereby suppressing MYCN-target genes. After inhibition of OCT4 binding, differentially downregulated transcripts were associated with high-open reading frame (ORF) dominance score, which is associated with the translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Furthermore, transcripts with a high-ORF dominance score were significantly associated with genes whose high expression is associated with a poor prognosis in NB. Because the ORF dominance score correlates with the translation efficiency of transcripts, our findings suggest that MYCN maintains the expression of transcripts with high translation efficiency, contributing to a poor prognosis in NB. In conclusion, the inhibition of OCT4 binding at the MYCN locus resulted in reduced MYCN activity, which in turn led to the downregulation of high-ORF dominance transcripts and subsequently induced caspase-2-mediated cell death in MYCN-amplified NB cells. Therefore, disruption of the OCT4 binding at the MYCN locus may serve as an effective therapeutic strategy for MYCN-amplified NB.</p