Tokuichi Kawaguchi

Whole-exome sequencing of human pancreatic cancers and characterization of genomic instability caused by MLH1 haploinsufficiency and complete deficiency

Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARID1C. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.

The selective continued linkage of centromeres from mitosis to interphase in the absence of mammalian separase

Separase is an evolutionarily conserved protease that is essential for chromosome segregation and cleaves cohesin Scc1/Rad21, which joins the sister chromatids together. Although mammalian separase also functions in chromosome segregation, our understanding of this process in mammals is still incomplete. We generated separase knockout mice, reporting an essential function for mammalian separase. Separase-deficient mouse embryonic fibroblasts exhibited severely restrained increases in cell number, polyploid chromosomes, and amplified centrosomes. Chromosome spreads demonstrated that multiple chromosomes connected to a centromeric region. Live observation demonstrated that the chromosomes of separase-deficient cells condensed, but failed to segregate, although subsequent cytokinesis and chromosome decondensation proceeded normally. These results establish that mammalian separase is essential for the separation of centromeres, but not of the arm regions of chromosomes. Other cell cycle events, such as mitotic exit, DNA replication, and centrosome duplication appear to occur normally. We also demonstrated that heterozygous separase-deficient cells exhibited severely restrained increases in cell number with apparently normal mitosis in the absence of securin, which is an inhibitory partner of separase.

Conversion of Human Colonic Adenoma Cells to Adenocarcinoma Cells Through Inflammation in Nude Mice

The roles of inflammation in the malignant progression of tumors during multistep carcinogenesis have been much discussed but remain to be elucidated. To determine the direct contribution of inflammation to colon carcinogenesis, we established a new model of progression of human colonic adenoma cells using a nude mouse; the progression is accelerated by coimplantation of a plastic plate. The FPCK-1–1 cell line, derived from a colonic polyp in a patient with familial adenomatous polyposis, is nontumorigenic when injected subcutaneously into nude mice in a cell suspension of up to 5 × 106 cells per mouse. However implantation of 1 × 105 FPCK-1–1 cells attached to a plastic plate induced first acute and then chronic inflammation, and formed progressively growing tumors that were histologically determined as moderately differentiated adenocarcinoma in 65% of mice. Moreover cell lines established from the growing tumors were found to be tumorigenic when injected into mice even without a plastic plate. The tumor arising from the adenoma cells implanted attached to a plastic plate was surrounded by highly proliferating fibrous stroma. This fibrous tissue was considered essential for malignant progression, rather than for attachment to the plastic plate substrate, because the tumors were formed after injection of FPCK-1–1 cells into the fibrous tissue from which the plastic plate had been removed before the cell injection. The conditioned medium (CM) obtained from the fibroblasts derived from a plastic plate-associated stromal tissue was found to contain factors that stimulated growth of FPCK-1–1 cells, but not of the derivative progressor cell lines. The factor was stable to heating and neuraminidase treatment, but labile to trypsin treatment. The main growth-potentiating activity was contained in the fraction larger than 100 kDa. In contrast, the activity to promote FPCK-1–1 cell growth was not present in the CM of subcutaneous fibroblasts from untreated nude mice or the fibroblast cell lines C3H10T 1/2 and NIH3T3. These results demonstrated that inflammation-associated stroma promoted the conversion of colonic adenoma cells to adenocarcinoma cells.

Epidermal growth factor‐dependent enhancement of invasiveness of squamous cell carcinoma of the breast

Factors that promote the aggressiveness of squamous cell carcinoma of the breast are not well understood. To examine the involvement of cell motility and the mechanism of this behavior, a squamous cell carcinoma cell line of the breast (HBC9) was established from a metastatic lymph node of a Japanese woman. HBC9 expressed epidermal growth factor receptor (EGFR), but was negative for Her2 or Her3.The invasive ability of HBC9 was compared with that of four breast ductal carcinoma cell lines by Matrigel invasion assay. EGF stimulation induced the formation of surface protrusions and cell migration in HBC9 cells, and significantly increased the number of cells migrating through the Matrigel. The invasive ability of HBC9 was compared with other cell lines of breast carcinoma; it was much greater than that of MCF‐7, BT474, or HBC5, but did not differ significantly from that of MDA‐MB‐231. Observation of the surface protrusions of HBC9 by confocal laser microscopy revealed co‐localization of Arp2 and N‐WASP with actin polymerization, detected by visualization with phalloidin, indicating that the protrusions induced by EGF were invadopodia. In HBC9 cells, cortactin also co‐localized with the N‐WASP/Arp2/3 complex in the protrusions. Immunohistochemistry of 12 cases of squamous cell carcinoma of the breast revealed expression of cortactin and EGFR in all of them, and this was confirmed by western blotting in two cases. These results suggest that EGF‐dependent enhancement of cell motility by formation of invadopodia associated with cortactin is a cause of the clinical aggressiveness of squamous cell carcinoma of the breast.

Identification of candidate predictive markers of anticancer drug sensitivity using a panel of human cancer cell lines.

We previously investigated the correlations between the expression of 9216 genes and various chemosensitivities in a panel of 39 human cancer cell lines1) and found that the expression levels of AKR1B1 and CTSH were correlated with sensitivity and resistance to multiple drugs, respectively. To validate these correlations, we investigated the expression of these two genes and the chemosensitivities in 12 additional gastric cancer cell lines. The expression of AKR1B1 in the additional cell lines exhibited significant correlations with sensitivities to 8 of the 23 drugs examined, while that of CTSH displayed a significant negative correlation with only one (MS‐247) of the 27 drugs examined. Their expressions were weakly correlated with sensitivity and resistance, respectively, to the remainder of the drugs. Moreover, when the 12 cell lines were divided into high‐expressing and low‐expressing groups, a comparison of these groups using Mann‐Whitneys U test revealed that high expression levels of AKR1B1 and CTSH were related to sensitivity to 21 of the drugs and resistance to 8 of the drugs, respectively. The present results suggest that AKR1B1 and CTSH may be good markers for prediction of sensitivity to certain drugs and that our panel of 39 cell lines has the potential to identify candidate predictive marker genes.

Fascin regulates chronic inflammation-related human colon carcinogenesis by inhibiting cell anoikis.

By a proteomics‐based approach, we identified an overexpression of fascin in colon adenocarcinoma cells (FPCKpP‐3) that developed from nontumorigenic human colonic adenoma cells (FPCK‐1–1) and were converted to tumorigenic by foreign‐body‐induced chronic inflammation in nude mice. Fascin overexpression was also observed in the tumors arising from rat intestinal epithelial cells (IEC 6) converted to tumorigenic in chronic inflammation which was induced in the same manner. Upregulation of fascin expression in FPCK‐1–1 cells by transfection with sense fascin cDNA converted the cells tumorigenic, whereas antisense fascin‐cDNA‐transfected FPCKpP‐3 cells reduced fascin expression and lost their tumor‐forming ability in vivo. The tumorigenic potential by fascin expression was consistent with their ability to survive and grow in the three‐dimensional multicellular spheroids. We found that resistance to anoikis (apoptotic cell death as a consequence of insufficient cell‐to‐substrate interactions), which is represented by the three‐dimensional growth of solid tumors in vivo, was regulated by fascin expression through caspase‐dependent apoptotic signals. From these, we demonstrate that fascin is a potent suppressor to caspase‐associated anoikis and accelerator of the conversion of colonic adenoma cells into adenocarcinoma cells by chronic inflammation.

Chronic inflammation-derived nitric oxide causes conversion of human colonic adenoma cells into adenocarcinoma cells.

It has been suggested that nitric oxide (NO) derived from chronically inflamed tissues is a cause of carcinogenesis. We herein demonstrated that administration of an inducible NO synthase inhibitor, aminoguanidine, significantly suppressed the tumorigenic conversion of human colonic adenoma (FPCK-1-1) cells into adenocarcinoma (FPCK/Inflam) cells accelerated by foreign body-induced chronic inflammation in nude mice. To determine whether NO directly promotes carcinogenesis, we exposed FPCK-1-1 cells continuously to chemically generated NO (FPCK/NO), and periodically examined their tumorigenicity. FPCK/NO cells formed tumors, whereas vehicle-treated cells (FPCK/NaOH) did not. We selected a tumorigenic population from FPCK/NO cells kept it in three-dimensional (3D) culture where in vivo-like multicellular spheroidal growth was expected. FPCK/Inflam cells developed large spheroids whereas FPCK/NO cells formed tiny but growing compact aggregates in 3D culture. Meanwhile, FPCK-1-1 and FPCK/NaOH cells underwent anoikis (apoptotic cell death consequential on insufficient cell-to-substrate interactions) through activation of caspase 3. The survived cells in the 3D culture (FPCK/NO/3D), which were derived from FPCK/NO cells, showed a similar tumor incidence to that of FPCK/Inflam cells. These results showed that NO was one of the causative factors for the acceleration of colon carcinogenesis, especially in the conversion from adenoma to adenocarcinoma in the chronic inflammatory environment.

Establishment and Characterization of an Epithelial Cell Line with Quasi‐normal Chromosomes from a Tubular Adenoma of a Familial Polyposis Coli Patient

An epithelial cell line designated FPCK‐1 has been established from a tubular adenoma developing in a male familial polyposis coli (FPC) patient. The FPCK‐1 cells grow very slowly with adundant mucus production and have been maintained stably for 3 years in culture. No growth was evident either in soft agar or nude mice, FPCK‐1 cells present a normal male karyotype and do not show loss of specific loci on chromosomes 5,17,18, and 22 which have been reported to be lost frequently in human colon carcinomas. The cells have neither a point mutation on codon 12 of K‐ras gene nor gene amplification of myc, c‐H‐ras, and/or c‐K‐ras genes. These results thus suggest the existence of hitherto unknown causative event(s) underlying adenoma development in FPC patients. The FPCK‐1 cell line should prove useful for further analytical investigation of the multiple steps involved in human colon carcinogenesis.

Ectopic chromosome around centrosome in metaphase cells as a marker of high-risk human papillomavirus-associated cervical intraepithelial neoplasias.

This report describes the appearance of ectopic chromosome around centrosome (ECAC) in metaphase cell nuclei of high‐risk HPV‐associated cervical neoplasms. ECAC are clearly visible on HE‐stained sections as a tiny (approx. 0.7 μ), round, dark structure or an aggregate of filamentous chromosome, often symmetrical at bilateral centrosomes. They appear in CINs from an early stage (CIN I), with the highest incidence in HPV16‐associated CIN II–III (75%), and are less common in HPV‐related invasive carcinomas (21%) and in lesions associated with high‐risk HPV types other than 16. Rates for ECAC‐positive nuclei in metaphase preparations (ECAC rate) for each lesion ranged 3.6–30%, the average being 14.5%. ECAC appeared very rarely in CINs associated with intermediate‐risk HPVs and was never encountered in HPV6/11‐induced condylomas or HPV‐unrelated neoplasms in other organs. ECAC may be morphologic evidence of HPV‐induced chromosomal instability working as a background mechanism in cervical carcinogenesis and also a useful marker for the histopathologic differentiation of high‐risk CINs.

Autonomous cure of damaged human intestinal epithelial cells by TLR2 and TLR4-dependent production of IL-22 in response to Spirulina polysaccharides

In order to analyze the damage of human epithelial cells, we used human quasi-normal FPCK-1-1 cells derived from a colonic polyp in a patient with familial adenomatous polyposis as a monolayer, which is co-cultured with peptidoglycan (PGN)-stimulated THP-1 cells. Co-cultured FPCK-1-1 cells showed a decreased transepithelial electrical resistance (TER) and the lower level of claudin-2. When Spirulina complex polysaccharides were added one day before the start of the co-culture, there was no decrease of TER and claudin-2 (early phase damage). In contrast, when Spirulina complex polysaccharides were added to FPCK-1-1 cells after the level of TER had decreased, there was no recovery at the level of claudin-2, though the TER level recovered (late phase damage). The mucosa reconstitution is suggested to be involved in the recovery from the damaged status. Interestingly, autonomous recovery of FPCK-1-1 cells from both the early and late phase damage requires the production of IL-22, because anti-IL-22 antibodies inhibited recovery in these cases. Antibodies against either TLR2 or TLR4 inhibited the production of IL-22 from FPCK-1-1 colon epithelial cells, suggesting that signals through TLR2 and TLR4 are necessary for autonomous recovery of FPCK-1-1 colon epithelial cells by producing IL-22. In conclusion, we have established a useful model for the study of intestinal damage and recovery using human colon epithelial cells and our data suggest that damage to human colon epithelial cells can, at least in part, be recovered by the autonomous production of IL-22 in response to Spirulina complex polysaccharides.