Scent test using Caenorhabditis elegans to screen for early-stage pancreatic cancer.

Although early detection and diagnosis are indispensable for improving the prognosis of patients with pancreatic cancer, both have yet to be achieved. Except for pancreatic cancer, other cancers have already been screened through scent tests using animals or microorganisms, including Caenorhabditis elegans. While such a method may greatly improve the prognosis of pancreatic cancer, no studies have investigated the same, mainly given the difficulty of collecting suitable samples from patients with early-stage pancreatic cancer. In this study, we organized a nationwide study group comprising high-volume centers throughout Japan to collect patients with very-early-stage pancreatic cancer (stage 0 or IA). We initially performed an open-label study involving 83 cases (stage 0-IV), with subsequent results showing significant differences after surgical removal in stage 0-IA (×10 dilution: p < 0.001; ×100 dilution: p < 0.001). Thereafter, a blinded study on 28 cases (11 patients with stage 0 or IA disease and 17 healthy volunteers) was conducted by comparing very-early-stage pancreatic cancer patients with healthy volunteers to determine whether C. elegans could detect the scent of cancer for the diagnosis of early-stage pancreatic cancer. Preoperative urine samples had a significantly higher chemotaxis index compared to postoperative samples in patients with pancreatic cancer [×10 dilution: p < 0.001, area under the receiver operating characteristic curve (AUC) = 0.845; ×100 dilution: p < 0.001, AUC = 0.820] and healthy volunteers (×10 dilution: p = 0.034; ×100 dilution: p = 0.088). Moreover, using the changes in preoperative and postoperative chemotaxis index, this method had a higher sensitivity for detecting early pancreatic cancer compared to existing diagnostic markers. The clinical application C. elegans for the early diagnosis of cancer can certainly be expected in the near future.

COVID-19 Drug Discovery Using Intensive Approaches.

Since the infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China during December 2019, the coronavirus disease 2019 (COVID-19) has spread on a global scale, causing the World Health Organization (WHO) to issue a warning. While novel vaccines and drugs that target SARS-CoV-2 are under development, this review provides information on therapeutics which are under clinical trials or are proposed to antagonize SARS-CoV-2. Based on the information gained from the responses to other RNA coronaviruses, including the strains that cause severe acute respiratory syndrome (SARS)-coronaviruses and Middle East respiratory syndrome (MERS), drug repurposing might be a viable strategy. Since several antiviral therapies can inhibit viral replication cycles or relieve symptoms, mechanisms unique to RNA viruses will be important for the clinical development of antivirals against SARS-CoV-2. Given that several currently marketed drugs may be efficient therapeutic agents for severe COVID-19 cases, they may be beneficial for future viral pandemics and other infections caused by RNA viruses when standard treatments are unavailable.

Embryonic MicroRNA-369 Controls Metabolic Splicing Factors and Urges Cellular Reprograming.

Noncoding microRNAs inhibit translation and lower the transcript stability of coding mRNA, however miR-369 s, in aberrant silencing genomic regions, stabilizes target proteins under cellular stress. We found that in vitro differentiation of embryonic stem cells led to chromatin methylation of histone H3K4 at the miR-369 region on chromosome 12qF in mice, which is expressed in embryonic cells and is critical for pluripotency. Proteomic analyses revealed that miR-369 stabilized translation of pyruvate kinase (Pkm2) splicing factors such as HNRNPA2B1. Overexpression of miR-369 stimulated Pkm2 splicing and enhanced induction of cellular reprogramming by induced pluripotent stem cell factors, whereas miR-369 knockdown resulted in suppression. Furthermore, immunoprecipitation analysis showed that the Argonaute complex contained the fragile X mental retardation-related protein 1 and HNRNPA2B1 in a miR-369-depedent manner. Our findings demonstrate a unique role of the embryonic miR-369-HNRNPA2B1 axis in controlling metabolic enzyme function, and suggest a novel pathway linking epigenetic, transcriptional, and metabolic control in cell reprogramming.

Pyruvate kinase M2, but not M1, allele maintains immature metabolic states of murine embryonic stem cells.

The M2 isoform of pyruvate kinase, the final rate-limiting enzyme of aerobic glycolysis, is expressed during embryonic development. In contrast, the M1 isoform is expressed in differentiated cells due to alternative splicing. Here we investigated murine embryonic stem cells (ESCs) with Pkm1 or Pkm2 knock-in alleles. Pkm1 allele knock-in resulted in excessive oxidative phosphorylation and induced the formation of cysteine-thiol disulfide-dependent complexes of forkhead box class-O (FOXO) transcription factors, which resulted in altered endoderm differentiation. In contrast, Pkm2 knock-in induced synthesis of a methylation-donor, S-adenosylmethionine, and increased unsaturated eicosanoid groups, which contributed to the redox control and maintenance of ESC undifferentiated status. Because PKM2 is also a critical enzyme for the cancer-specific Warburg effect, our results demonstrate an important role for the Pkm2 allele in establishing intracellular redox conditions and modulating PKM1-dependent oxidative phosphorylation events to achieve an appropriate ESC differentiation program.

SCGB2A1 is a novel prognostic marker for colorectal cancer associated with chemoresistance and radioresistance.

We recently showed that liver metastatic tissue from patients with colorectal cancer (CRC) was a useful model for identifying novel, hypoxia-inducible genes and prognostic markers. We showed that the expression of secretoglobin, family 2A, member 1 (SCGB2A1) was a potential prognostic factor for CRC. Here, we further evaluated the prognostic impact and function of SCGB2A1 in 222 patients with CRC. The impact of SCGB2A1 expression on disease-free survival (DFS) and overall survival (OS) was assessed with mRNA expression profiling. The function of SCGB2A1 was analyzed by evaluating mRNA expression profiles in cells derived from patients with CRC and by testing the effects of transfecting SCGB2A1 into different CRC-derived cell lines. We evaluated the effects of SCGB2A1 on proliferation, chemosensitivity, radiation sensitivity and sphere formation. Univariate and multivariate analyses indicated that the expression of SCGB2A1 was an independent prognostic factor for CRC (p<0.05), together with lymph node metastasis (p<0.05). Enforced expression of SCGB2A1 in CRC-derived cell lines promoted proliferation (DLD1, SW480 and LoVo cells; p<0.05), decreased chemosensitivity to 5-fluorouracil and oxaliplatin (DLD1 and SW480 cell lines; p<0.05), and significantly increased the viability of irradiated cells (DLD1, SW480 and LoVo cell lines; p<0.05). SCGB2A1 expression was also correlated to cancer stemness-related genes (Wnt, Zeb1 and Twist). Consistent with this correlation, SCGB2A1 expressing cells (SW480) showed increased sphere formation (p<0.05). These results indicated that SCGB2A1 represented a novel, prognostic factor for CRC, and that expression of SCGB2A1 correlated with chemoresistance, radioresistance and cancer cell stemness.

[Cancer stem cells depend on their own niche].

It is considered that cancer stem cells have the same characteristics as normal stem cells, such as drug-resistance, self-renewal, differentiation, and tissue-formation. Normal stem cells depend on their surroundings, a niche. Cancer stem cells may also depend on their own niche. Because cancer stem cells are resistant to present remedies, it is important to find a remedy targeting cancer stem cells. The remedy must not only target cancer stem cells themselves but also target the niche surrounding the cancer stem cells.

Adipose-derived mesenchymal stem cells and regenerative medicine.

Adipose tissue-derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic ß-cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow-derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.

Depletion of JARID1B induces cellular senescence in human colorectal cancer.

The global incidence of colorectal cancer (CRC) is increasing. Although there are emerging epigenetic factors that contribute to the occurrence, development and metastasis of CRC, the biological significance of epigenetic molecular regulation in different subpopulations such as cancer stem cells remains to be elucidated. In this study, we investigated the functional roles of the H3K4 demethylase, jumonji, AT rich interactive domain 1B (JARID1B), an epigenetic factor required for the continuous cell growth of melanomas, in CRC. We found that CD44(+)/aldehyde dehydrogenase (ALDH)(+) slowly proliferating immature CRC stem cell populations expressed relatively low levels of JARID1B and the differentiation marker, CD20, as well as relatively high levels of the tumor suppressor, p16/INK4A. Of note, lentiviral­mediated continuous JARID1B depletion resulted in the loss of epithelial differentiation and suppressed CRC cell growth, which was associated with the induction of phosphorylation by the c­Jun N­terminal kinase (Jnk/Sapk) and senescence­associated ß­galactosidase activity. Moreover, green fluorescent­labeled cell tracking indicated that JARID1B­positive CRC cells had greater tumorigenicity than JARID1B­negative CRC cells after their subcutaneous inoculation into immunodeficient mice, although JARID1B­negative CRC cells resumed normal growth after a month, suggesting that continuous JARID1B inhibition is necessary for tumor eradication. Thus, JARID1B plays a role in CRC maintenance. JARID1B may be a novel molecular target for therapy­resistant cancer cells by the induction of cellular senescence.

Eradication of therapy-resistant cancer cells in gastrointestinal organs.

Early stages of cancer are curable by surgical removal of the primary lesions, however, more advanced cases are often refractory to therapeutic approaches and are more commonly life-threatening, primarily due to cancer metastasis in gastrointestinal cancers. Such biological events are collectively characterized as tumor heterogeneity, the cause of which is the existence of cancer stem cells. To improve cancer survival, therapy-resistant cancer cells should be eradicated. To this end, recent rapid progress in medical science, such as innovative medical technologies including cancer reprogramming, RNA pharmacology and drug delivery systems, all of which effectively target cancer stem cells, has facilitated this objective.

Jumonji/Arid1b (Jarid1b) protein modulates human esophageal cancer cell growth.

Although esophageal cancer is highly heterogeneous and the involvement of epigenetic regulation of cancer stem cells is highly suspected, the biological significance of epigenetically modified molecules that regulate different subpopulations remains to be firmly established. Using esophageal cancer cells, we investigated the functional roles of the H3K4 demethylase Jumonji/Arid1b (Jarid1b) (Kdm5b/Plu-1/Rbp2-h1), an epigenetic factor that is required for continuous cell growth in melanoma. JARID1B knockdown resulted in the suppression of esophageal cancer cell growth, sphere formation and invasion ability and was associated with loss of epithelial marker expression. However, these inhibitory effects observed on tumor formation were reverted subsequent to subcutaneous inoculation of these cells into immune-deficient mice. These results indicated that JARID1B plays a role in maintaining cancer stem cells in the esophagus and justifies the rationale for studying the effects of continuous inhibition of this epigenetic factor in esophageal cancer.

microRNA-based cancer cell reprogramming technology.

Epigenetic modifications play crucial roles in cancer initiation and development. Complete reprogramming can be achieved through the introduction of defined biological factors such as Oct4, Sox2, Klf4, and cMyc into mouse and human fibroblasts. Introduction of these transcription factors resulted in the modification of malignant phenotype behavior. Recent studies have shown that human and mouse somatic cells can be reprogrammed to become induced pluripotent stem cells using forced expression of microRNAs, which completely eliminates the need for ectopic protein expression. Considering the usefulness of RNA molecules, microRNA-based reprogramming technology may have future applications in regenerative and cancer medicine.

Genotoxic therapy stimulates error-prone DNA repair in dormant hepatocellular cancer stem cells.

Previous studies have described distinct dormant and proliferating populations of cancer stem cells in hepatocellular carcinoma. The CD13 protein is involved in the scavenging of reactive oxygen species through the glutathione reductase pathway and is associated with resistance to chemotherapy. Whereas CD13(-) proliferating cancer stem cells are sensitive to chemotherapy, CD13(+) dormant cancer stem cells are associated with the development of resistance to chemotherapy. CD13(+) cells in hypoxic areas of the tumour survive chemotherapy, leading to subsequent disease relapse and metastasis. Whether CD13(+) dormant cells simply resume proliferation following therapy or whether they also acquire greater malignant potential, remains unknown. The mechanisms involved also remain unclear. In the present study, we investigated the repair of DNA damage in CD13(+) dormant and CD13(-) proliferating cells. Total RNA was extracted from tissues, and quantitative real-time polymerase chain reaction (PCR) was performed for specific genes and GAPDH following PCR. Products were then subjected to a temperature gradient of 55-95°C with continuous fluorescence monitoring to generate a melting curve. Cells were incubated with primary antibodies, washed twice, incubated with fluorescent-labelled secondary antibodies for 30 min on ice and analyzed by flow cytometry. The results revealed that the repair of DNA damage in CD13(+) dormant cells occurs predominantly through non-homologous end-joining, a repair process that is error-prone, whereas CD13(-) proliferating cells primarily utilise high-fidelity homologous recombination for DNA repair. These data indicate that not only is dormancy a protective mechanism for cancer stem cells to survive therapy, but it also enhances the generation and accumulation of mutations following DNA damage. Therefore, the CD13(+) dormant cancer stem cells must be eradicated fully to achieve complete remission of cancer.

Transcriptomic study of dormant gastrointestinal cancer stem cells.

We previously discovered the coexistence of dormant and proliferating cancer stem cells (CSCs) in gastrointestinal cancer, which leads to chemoradiation resistance. CD13-/CD90+ proliferating liver CSCs are sensitive to chemotherapy, and CD13+/CD90- dormant CSCs have a limited proliferation ability, survive in hypoxic areas with reduced oxidative stress, and relapse and metastasize to other organs. In such CD13+ dormant cells, non-homologous end-joining, an error-prone repair mechanism, is dominant after DNA damage, whereas high-fidelity homologous recombination is apparent in CD13- proliferating cells, suggesting the significance of dormancy as an essential protective mechanism of therapy resistance. However, this mechanism may also play a role in the generation and accumulation of heterogeneity during cancer progression, although the exact mechanism remains to be understood. Through transcriptomic study, we elucidated the underlying epigenetic mechanism for malignant behavior of dormant CSCs, i.e., simultaneous activation of several pathways including EZH2- and TP53-related proteins in response to microRNA101, suggesting that a pharmacogenomic approach would open an era to novel molecular targeting cancer therapy.

Dicer 1, ribonuclease type III modulates a reprogramming effect in colorectal cancer cells.

Complete cell reprogramming can be achieved by the introduction of specific transcription factors, Oct4 [also known as POU class 5 homeobox 1 (Pou5f1)]; sex-determining region Y (SRY)-box 2 (Sox2); Kruppel-like factor 4 (Klf4); and myelocytomatosis viral oncogene homolog (c-Myc), into terminally differentiated mouse somatic fibroblasts. This reprogramming process may be accelerated or suppressed by various factors, including microRNAs (miRNAs). Introduction of these transcription factors or miRNAs considerably modifies the malignant phenotype of cancer cells. We studied the effect of introducing these transcription factors into two distinct colorectal cancer (CRC) cell lines, HCT116 and DLD-1, in the presence and absence of Dicer 1, ribonuclease type III (Dicer1), a critical miRNA processing enzyme. We assessed cell reprogramming based on the number of cells exhibiting alkaline phosphatase staining and an increase in embryonic stem cell-like gene expression, indicating the return of cells to an immature state. Dicer1-deficient CRC cells showed a reduced number of alkaline phosphatase-positive reprogrammed cells than wild-type (WT) cells. Before reprogramming, endogenous expression of an immature carbohydrate epitope, TRA-1-60, was high in Dicer1-deficient CRC cells, whereas after reprogramming, the expression of this epitope was increased in Dicer1-sufficient more than in Dicer1-deficient CRC cells. Our data demonstrate the critical role of miRNAs in the reprogramming process and determination of a differentiated phenotype of CRC cells.

Hypoxia and TP53 deficiency for induced pluripotent stem cell-like properties in gastrointestinal cancer.

Induced pluripotent stem (iPS)-like cancer cells (iPC) by the introduction of defined transcription factors reduce the prevalence of the malignant phenotype of digestive system cancer cells, but the induction efficiency is low. The role of hypoxia and TP53 deficiency in iPC cell generation remain unclear. Cellular reprogramming was performed by retroviral infection with OCT3/4, SOX2, KLF4 and c-MYC of wild-type HCT116 colorectal cancer cells and mutant TP53-deficient HCT116 cells. Cells were cultured in normoxia (21% O2) or hypoxia (5% O2) for 30 days after transduction, and the response to hypoxia and comparison of cellular proliferation, invasion and tumourigenesis before and after iPC cell generation were studied. iPC cell generation from wild-type HCT116 cells in hypoxia was approximately 4-times greater than in normoxia (p<0.05), and TP53 deficiency increased conversion efficiency significantly in normoxia (p<0.05). Significant involvement of hypoxia-inducible factors was observed in an immature carbohydrate epitope, Tra-1-60+, colony formation. Generated iPC cells exhibited multi-differentiation potential. Although the iPC cells in hypoxia exhibited reduced proliferation, invasiveness and tumourigenicity, TP53 deficiency in iPC cells resulted in higher tumourigenicity than in wild-type cells. Both hypoxia and TP53 deficiency increase iPC cell generation. TP53 deficiency can also result in deleterious mutations, whereas hypoxia may impact molecular targets of epigenome normalisation.

Reprogramming of gastrointestinal cancer cells.

Cell reprogramming reverts cells to multipotent, preprogrammed states by re-establishing epigenetic markers. It can also induce considerable malignant phenotype modification. Because key events in cancer relapse and metastasis, including epithelial-mesenchymal transition phenotypes, are regulated primarily by reversible and transient epigenetic modifications rather than the accumulation of irreversible and stable genetic abnormalities, studying dynamic mechanisms regulating these biological processes is important. Transcription factors for induced pluripotent stem cells and non-coding microRNAs allow pluripotent phenotype induction. We present the current knowledge of the possible applications of cell reprogramming in reducing aggressive phenotype expression, which can induce tumor cell hibernation and maintain appropriate phenotypes, thereby minimizing relapse and metastasis after surgical resection of gastrointestinal cancer.