Chondroitin sulfate proteoglycan 4: An attractive target for antibody-based immunotherapy.

Multifunctional molecules involved in tumor progression and metastasis have been identified as valuable targets for immunotherapy. Among these, chondroitin sulfate proteoglycan 4 (CSPG4), a significant tumor cell membrane-bound proteoglycan, has emerged as a promising target, especially in light of advances in chimeric antigen receptor (CAR) T-cell therapy. The profound bioactivity of CSPG4 and its role in pivotal processes such as tumor proliferation, migration, and neoangiogenesis underline its therapeutic potential. We reviewed the molecular intricacies of CSPG4, its functional attributes within tumor cells, and the latest clinical-translational advances targeting it. Strategies such as blocking monoclonal antibodies, conjugate therapies, bispecific antibodies, small-molecule inhibitors, CAR T-cell therapies, trispecific killer engagers, and ribonucleic acid vaccines against CSPG4 were assessed. CSPG4 overexpression in diverse tumors and its correlation with adverse prognostic outcomes emphasize its significance in cancer biology. These findings suggest that targeting CSPG4 offers a promising avenue for future cancer therapy, with potential synergistic effects when combined with existing treatments.

Novel chimeric antigen receptor-expressing T cells targeting the malignant mesothelioma-specific antigen sialylated HEG1.

The selection of highly specific target antigens is critical for the development of clinically efficient and safe chimeric antigen receptors (CARs). In search of diagnostic marker for malignant mesothelioma (MM), we have established SKM9-2 monoclonal antibody (mAb) which recognizes a MM-specific molecule, sialylated Protein HEG homolog 1 (HEG1), with high specificity and sensitivity. In this study, to develop a novel therapeutic approach against MM, we generated SKM9-2 mAb-derived CARs that included the CD28 (SKM-28z) or 4-1BB (SKM-BBz) costimulatory domain. SKM-28z CAR-T cells showed continuous growth and enhanced Tim-3, LAG-3, and PD-1 expression in vitro, which might be induced by tonic signaling caused by self-activation; however, these phenotypes were not observed in SKM-BBz CAR-T cells. In addition, SKM-BBz CAR-T cells exhibited slightly stronger in vitro killing activity against MM cell lines than SKM-28z CAR-T cells. More importantly, only SKM-BBz CAR-T cells, but not SKM-28z CAR-T cells, significantly inhibited tumor growth in vivo in a MM cell line xenograft mouse model. Gene expression profiling and reporter assays revealed differential signaling pathway activation; in particular, SKM-BBz CAR-T cells exhibited enhanced NF-kB signaling and reduced NFAT activation. In addition, SKM-BBz CAR-T cells showed upregulation of early memory markers, such as TCF7 and CCR7, as well as downregulation of pro-apoptotic proteins, such as BAK1 and BID, which may be associated with phenotypical and functional differences between SKM-BBz and SKM-28z CAR-T cells. In conclusion, we developed novel SKM9-2-derived CAR-T cells with the 4-1BB costimulatory domain, which could provide a promising therapeutic approach against refractory MM.

Medical application of the monoclonal antibody SKM9-2 against sialylated HEG1, a new precision marker for malignant mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive tumor of the pleural cavity. Pathologically distinguishing MPM from other pleural lesions is often difficult. We searched for marker antigens to facilitate the pathological diagnosis of MPM and found useful markers for the pathological detection of malignant mesothelioma. Among them, the anti-mesothelioma monoclonal antibody SKM9-2, which was isolated as a clone binding to specimens of MPM (but not to specimens of lung adenocarcinoma) by immunohistochemical screening, showed higher specificity and sensitivity than traditional mesothelioma markers. SKM9-2 recognizes both sialylated O-glycans and peptide sequences in HEG1, and its glycan modifications are specific to mesothelioma. New effective treatments for MPM are needed because the prognosis of patients with MPM is usually poor. SKM9-2 can be used as a seed for next-generation antibody drugs with strong cytotoxic activities. In this review, we have summarized our research on antibody development for MPM diagnosis and treatment.

Therapeutic siRNA targeting the cancer cell stemness regulator PRDI-BF1 and RIZ domain zinc finger protein 14.

PRDI-BF1 and RIZ (PR) domain zinc finger protein 14 (PRDM14), first reported in 2007 to be overexpressed in breast cancer, plays an important role in breast cancer proliferation. Subsequent studies reported that PRDM14 is expressed in embryonic stem cells, primordial germ cells, and various cancers. PRDM14 was reported to confer stemness properties to cancer cells. These properties induce cancer initiation, cancer progression, therapeutic resistance, distant metastasis, and recurrence in refractory tumors. Therefore, PRDM14 may be an ideal therapeutic target for various types of tumors. Silencing PRDM14 expression using PRDM14-specific siRNA delivered through an innovative intravenous drug delivery system reduced the size of inoculated tumors, incidence of distant metastases, and increased overall survival in nude mice without causing adverse effects. Therapeutic siRNA targeting PRDM14 is now being evaluated in a human phase I clinical trial for patients with refractory breast cancer, including triple-negative breast cancer.

Antitumoral RNA-targeted oligonucleotide therapeutics: The third pillar after small molecule inhibitors and antibodies.

Oligonucleotide therapeutics, drugs consisting of 10-50 nucleotide-long single- or double-stranded DNA or RNA molecules that can bind to specific DNA or RNA sequences or proteins, include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), aptamers, and decoys. These oligonucleotide therapeutics could potentially become the third pillar of drug development. In particular, ASOs and siRNAs are advanced tools that are widely used to silence gene expression. They are used in clinical trials, as they have high specificity for target mRNAs and non-coding RNAs and limited toxicity. However, their clinical application remains challenging. Although chemotherapy has benefits, it has severe adverse effects in many patients. Therefore, new modalities for targeted molecular therapy against tumors, including oligonucleotide therapeutics, are required, and they should be compatible with diagnosis using next-generation sequencing. This review provides an overview of the therapeutic uses of ASOs, siRNAs, and miRNAs in clinical studies on malignant tumors. Understanding previous research and development will help in developing novel oligonucleotide therapeutics against malignant tumors.

Membranous HEG1 expression is a useful marker in the differential diagnosis of epithelioid and biphasic malignant mesothelioma versus carcinomas.

Sialylated HEG1 has been reported as a highly specific and sensitive mesothelioma marker but a comprehensive evaluation of its expression in carcinomas in different organs, various sarcomas and reactive mesothelial proliferations has not been reported. The aim of this study was to evaluate the clinical applicability of HEG1 as a marker in the diagnosis of mesothelioma. HEG1 immunoreactivity was evaluated in whole sections of 122 mesotheliomas, 75 pulmonary carcinomas, 55 other carcinomas, 16 mesenchymal tumors, and 24 reactive mesothelial proliferations and in tissue microarrays containing 70 epithelioid (EM), 36 biphasic (BM), and 2 sarcomatoid mesotheliomas (SM). In whole sections and tissue microarrays, respectively, membranous HEG1 was expressed in 93.0% and 85.5% of EM, 81.3% and 69.4% of BM, 0% and 0% of SM. HEG1 was not expressed in pulmonary adenocarcinomas. HEG1 was expressed as cytoplasmic immunoreactivity in pulmonary squamous cell carcinomas (21.7%). Membranous HEG1 staining was seen in ovarian carcinomas (66.7%), thyroid carcinomas (100%), reactive conditions (16.7%), and mesenchymal tumors (18.8%). The sensitivity of membranous HEG1 expression to distinguish EM/BM from all carcinomas was 88.8%. The specificity for the differential diagnosis between EM/BM and all carcinomas and pulmonary carcinomas was 92.3% and 98.7%, respectively.

Treatment of primary and metastatic breast and pancreatic tumors upon intravenous delivery of a PRDM14-specific chimeric siRNA/nanocarrier complex.

PRDM14 is highly expressed in several cancers but is not detected in normal tissues. It confers cancer stem cell-like properties, including chemoresistance and distant metastasis, to cancer cells. Herein, we aimed to develop a highly effective therapy against advanced stage cancer based on intravenously delivered PRDM14-targeted siRNA. First, we examined PRDM14 expression and gene amplification in breast and pancreatic tumors and cell lines. PRDM14 was expressed in breast cancer, including the triple-negative subtype, and pancreatic cancer. PRDM14 was amplified in 23.8% of patients with PRDM14+ breast cancer. Next, we investigated the inoculated tumor growth and distant metastasis following PRDM14 depletion by administering mice with PRDM14-specific chimeric siRNA combined with a novel branched PEGylated poly-L-ornithine (PLO)-based intravenous drug delivery system, designated PRDM14 unit polyion complex (uPIC) (n = 6/group). Inhibition of PRDM14 expression with PRDM14 uPIC by systemic intravenous injection effectively reduced tumor size and metastasis in vivo, thereby improving survival. Finally, pharmacokinetic/toxicokinetic analyses were performed on PRDM14 uPIC, which was intravenously administered to rats (n = 10-15/group) and cynomolgus monkeys (n = 3-5/group), twice weekly for 4 weeks. This revealed that PRDM14 uPIC was relatively nontoxic and the siRNA exposure in serum was greater than that predicted by the administered dose ratio when delivered as a uPIC. Taken together, our study indicated that PRDM14 uPIC is highly effective in suppressing malignant features of solid cancers and does not cause severe toxicity, making it a promising therapeutic agent for cancer treatment.

HEG1, BAP1, and MTAP are useful in cytologic diagnosis of malignant mesothelioma with effusion.

BACKGROUND: The specificity and sensitivity of HEG1 for malignant mesothelioma (MM) is high. The use of BAP1/MTAP immunohistochemistry (IHC) is recommended to separate benign and malignant mesothelial proliferations. We determined how ancillary techniques can be used for the cytological diagnosis of MM with effusion. METHODS: Cell blocks from effusions from cases with MM, reactive mesothelial cells (RMCs), and carcinomas were analyzed by IHC with HEG1, BAP1, and MTAP and with homozygous deletion (HD) of CDKN2A by fluorescence in situ hybridization. Staining scores were calculated for IHC by adding the number of categories for the staining intensity and the staining extension. RESULTS: HEG1 was positive in all (41/41) MMs, but negative in carcinomas, except for ovarian carcinomas. Overall 76.9% (20/26) of RMCs and 28.6% (6/21) of ovarian carcinomas expressed HEG1. BAP1 loss was found in 71.1% of MMs, but none was found in RMCs. MTAP loss was found in 76.2% of MMs, but none was found in RMCs. 73.9% of MMs harbored HD of CDKN2A. There was concordance between loss of MTAP and HD of CDKN2A in 95% of MMs. CONCLUSION: HEG1 is a good marker for mesothelial differentiation in effusion cytology. HD of CDKN2A is frequently observed in cell blocks from effusions of MMs, and MTAP IHC may act as a surrogate for HD of CDKN2A. Cell block analysis is recommended for effusions of unknown origins with the following methods: IHC with HEG1 and claudin 4 to validate the mesothelial origin, followed by BAP1 and MTAP IHC to confirm malignancy.

Microsatellite instability in cancer: a novel landscape for diagnostic and therapeutic approach.

Defective DNA mismatch repair creates a strong mutator phenotype, recognized as microsatellite instability (MSI). Various next-generation sequencing-based methods for evaluating cancer MSI status have been established, and NGS-based studies have thoroughly described MSI-driven tumorigenesis. Accordingly, high-frequency MSI (MSI-H) has been detected in 81 tumor types, including those in which MSI was previously underrated. The findings have increased the use of immunotherapy, which is assumed to be efficient in tumors having a high mutation burden and/or neoantigen load. In MSI tumorigenesis, positively and negatively selected driver gene mutations have been characterized in colorectal cancers. Recent advancements in genome-wide studies of MSI-H cancers have developed novel diagnostic and therapeutic approaches, including CXCR2 inhibitor, a synthetic lethal therapy targeting the Werner gene and inhibition of nonsense-mediated mRNA decay. MSI is a predictive marker for chemotherapy as well as immunotherapy. Thus, analyses of MSI status and MSI-related alterations in cancers are clinically relevant. We present an update on MSI-driven tumorigenesis, focusing on a novel landscape of diagnostic and therapeutic approaches.

An updated review of microsatellite instability in the era of next-generation sequencing and precision medicine.

Deficient DNA mismatch repair causes a robust mutator phenotype known as microsatellite instability (MSI). MSI is a feature of Lynch syndrome-related cancers and is also found in approximately 15% of sporadic gastric, colorectal, and endometrial cancers. Epigenetic inactivation of the MLH1 gene is often associated with sporadic MSI cancers. Recent next-generation sequencing (NGS)-based analyses have comprehensively characterized MSI-positive (MSI+) cancers, and several approaches for detecting the MSI phenotype of tumors using NGS have been developed. The FDA has recently granted accelerated approval to an anti-PD-1 antibody, pembrolizumab, for use in pediatric and adult patients with MSI+ solid tumors. Genome-wide analyses using NGS have revealed that hypermutation defined as >10 somatic mutations per megabase appears to be more prevalent than previously estimated, affecting approximately 17% of adult cancers. These results potentially expand the use of immunotherapy, which is thought to be effective in cancers with an increased mutational burden. Therefore, evaluation of MSI and MSI-associated molecular changes in tumors has emerged as clinically important. MSI is a valuable diagnostic marker of Lynch syndrome and a potential predictive marker for chemotherapy and immunotherapy efficacy. Here, we provide an update on MSI-associated cancers, focusing on findings obtained by genome-wide analyses using NGS, and the predictive role of MSI in immune checkpoint immunotherapy.

Immunohistological analysis of pancreatic carcinoma after vaccination with survivin 2B peptide: Analysis of an autopsy series.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer by providing new options in addition to existing therapies. However, peptide vaccination therapies still represent an attractive approach, because of the antigen specificity. We identified survivin 2B peptide (SVN-2B), a 9-mer antigenic peptide encoded by survivin, and an SVN-2B peptide vaccine-based phase II randomized clinical trial targeting unresectable and refractory pancreatic carcinoma was undertaken. The SVN-2B peptide vaccine did not have any statistically significant clinical benefits in that study. Therefore, we undertook an autopsy study to analyze the immune status of the pancreatic cancer lesions at the histological level. Autopsies were carried out in 13 patients who had died of pancreatic cancer, including 7 who had received SVN-2B peptide vaccination and 6 who had not, as negative controls. The expression of immune-related molecules was analyzed by immunohistochemical staining. Cytotoxic T lymphocytes were analyzed by tetramer staining and enzyme-linked immunospot assay. Histological analysis revealed dense infiltration of CD8+ T cells in some lesions in patients who had received the SVN-2B peptide vaccine. A high rate of programmed cell death ligand 1 expression in cancer cells was observed in these cases, indicating that CTLs were induced by SVN-2B peptide vaccination and had infiltrated the lesions. The lack of a significant antitumor effect was most likely attributable to the expression of immune checkpoint molecules. These findings suggest that the combination of a tumor-specific peptide vaccine and an ICI might be a promising approach to the treatment of pancreatic carcinoma in the future.

Randomized phase II trial of survivin 2B peptide vaccination for patients with HLA-A24-positive pancreatic adenocarcinoma.

The prognosis of advanced pancreatic adenocarcinoma is still extremely poor. This study sought to determine the efficacy of, and immunological response to, peptide vaccination therapy in patients with this disease. In this multicenter randomized phase II study, patients with advanced pancreatic adenocarcinoma after gemcitabine and/or tegafur/gimeracil/oteracil were randomly assigned to 3 groups that each received a 2-step treatment course. In Step 1, the groups received treatments of: (i) survivin 2B peptide (SVN-2B) plus interferon-ß (IFNß); (ii) SVN-2B only; or (iii) placebo until the patients show progression. In Step 2, all patients who consented to participate received 4 treatments with SVN-2B plus IFNß. The primary endpoint was progression-free survival (PFS) after initiation of Step 1 treatment. Secondary endpoints included immunological effects assessed by analysis of PBMCs after Step 1. Eighty-three patients were randomly assigned to receive SVN-2B plus IFNß (n = 30), SVN-2B (n = 34), or placebo (n = 19). No significant improvement in PFS was observed. Survivin 2B-specific CTLs were found to be increased in the SVN-2B plus IFNß group by tetramer assay. Among patients who participated in Step 2, those who had received SVN-2B plus IFNß in Step 1 showed better overall survival compared with those who had received placebo in Step 1. Patients vaccinated with SVN-2B plus IFNß did not have improved PFS, but showed significant immunological reaction after vaccination. Subgroup analysis suggested that a longer SVN-2B plus IFNß vaccination protocol might confer survival benefit. (Clinical trial registration number: UMIN 000012146).

Silencing PRDM14 via Oligonucleotide Therapeutics Suppresses Tumorigenicity and Metastasis of Breast Cancer.

The PRDI-BF1 and RIZ (PR) domain zinc finger protein 14 (PRDM14) is upregulated in approximately 60% of breast cancers, some of which exhibit gene amplification. In contrast, PRDM14 is not expressed in normal, and differentiated tissues. PRDM14+ breast cancer cells are resistant to chemotherapy drugs, are tumorigenic, and metastasize to the lungs. It is commonly assumed that genes that are overexpressed in cancers, such as PRDM14, are effective targets for new therapies that specifically abrogate the expression of these genes. RNA interference of PRDM14, a gene expressed by breast cancer cells, reduced the size of tumors and lung metastases in nude mice. In this chapter, we introduce the concept and methods to develop and apply systematically injected small interfering RNA therapy for breast cancer models in vivo.

PRDM14 is overexpressed in chronic pancreatitis prior to pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer that is typically diagnosed at a later stage with metastases and is difficult to treat. Therefore, investigating the mechanism of PDAC initiation is important to aid early-stage cancer detection. PRDM14 is a transcription factor that maintains pluripotency in embryonic stem cells and is overexpressed in several cancers. We previously reported that PRDM14 is overexpressed and regulates cancer stem-like phenotypes in PDAC, and herein, we assess whether PRDM14 expression increases prior to tumorigenesis. Through immunohistochemistry analyses of clinical tissues, we detected PRDM14-positive cells in precursor pancreatic intraepithelial neoplasia and chronic pancreatitis, which is a risk factor for PDAC, lesions. PRDM14 staining in chronic pancreatitis was as high as that in PDAC and cancer adjacent tissues. We induced pancreatitis in mouse models by cerulein injection, and observed that PRDM14 expression increased in chronic pancreatitis models but not in control or acute pancreatitis mice. Moreover, cerulein treatment increased PRDM14 expression in PK-1 and AsPC-1 pancreatic cancer cell lines. Our results suggest that inflammation increases the expression of PRDM14, which regulates cancer stem-like phenotypes, and this occurs prior to PDAC initiation and progression.

Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1.

The anti-mesothelioma mAb SKM9-2 recognizes the sialylated protein HEG homolog 1 (HEG1). HEG1 is a 400 kDa mucin-like membrane protein found on mesothelioma. SKM9-2 can detect mesothelioma more specifically and sensitively than other antibodies against current mesothelioma markers; therefore, SKM9-2 would be likely useful for the precise detection and diagnosis of malignant mesothelioma. In the present study, we investigated the epitope of SKM9-2. We analyzed the binding of SKM9-2 to truncated HEG1 and candidate epitope-fused glycosylphosphatidylinositol-anchor proteins. The epitope of SKM9-2 was identified as an O-glycosylated region, 893-SKSPSLVSLPT-903, in HEG1. An alanine scanning assay of the epitope showed that SKM9-2 bound to a simple epitope in HEG1, and the SKxPSxVS sequence within the epitope was essential for SKM9-2 recognition. Mass spectrometry analysis and lectin binding analysis of soluble epitope peptides indicated that the SKM9-2 epitope, in which Ser897 was not glycosylated, contained two disialylated core 1 O-linked glycan-modified serine residues, Ser893 and Ser900. Neuraminidase treatment analysis also confirmed that the epitope in mesothelioma cells contained a similar glycan modification. The specific detection of mesothelioma with SKM9-2 can thus be performed by the recognition of sialylated glycan modification in the specific region of HEG1.

PRDM14, a Zinc Finger Protein, Regulates Cancer Stemness.

PRDI-BF1 and RIZ homology (PR) domain zinc finger protein 14 (PRDM14) contains a PR domain related to the SET methyltransferase domain and zinc finger motifs. PRDM14 maintains stemness in embryonic stem cells and primordial germ cells via epigenetic mechanisms. PRDM14, however, is not expressed in normal differentiated tissues. We and other groups previously reported that PRDM14 expression is markedly higher in some types of cancers compared to the corresponding normal tissues. PRDM14 confers stem cell-like characteristics upon cancer cells, such as sphere formation, dye efflux, chemotherapy resistance, proliferation, and distant metastasis. Cancer stem cells (CSCs) are thought to be responsible for tumor initiation, drug and radiation resistance, invasive growth, metastasis, and tumor relapse, which are the primary causes of cancer-related deaths. Because CSCs are also thought to be resistant to conventional therapies, an effective and novel therapeutic approach for CSCs is imperative.RNAi silencing of PRDM14 expressed by breast and pancreatic cancer cells reduced tumor size and distant metastasis of these cells in nude mice. Inhibition of PRDM14 expression by cancer cells may be an effective and radical therapy for solid cancers. In this chapter, we discuss methods for studying CSC-like properties in cancer cells and describe the use of siRNA with a drug delivery system by systemic injection in vivo.

Prevalence of myofascial pain syndrome in patients with incurable cancer.

BACKGROUND: Myofascial pain syndrome (MPS) is a condition that involves skeletal muscles. It is caused by overload or disuse of muscles and is characterized by extreme tenderness in the muscles with taut bands. Treatment for MPS is different from that for cancer-related pain. Cancer patients have many factors that cause restriction of body movement and posture. Although cancer patients appear to demonstrate risk factors for MPS, its prevalence has not been reported in patients with incurable cancer. This study was conducted to investigate the prevalence of MPS in patients with incurable cancer. METHODS: A retrospective chart review. The data for patients with incurable cancer who received palliative care at our department between September 2015 and March 2016 were investigated. We examined the prevalence of MPS, which was diagnosed on the basis of the Rivers criteria (RC) and Simons criteria (SC). We also examined the following factors associated with MPS: performance status (PS), use of medical devices, and primary cancer sites. The primary outcome was the prevalence of MPS based on RC. Secondary outcomes included the prevalence of MPS based on SC and the relationship between MPS and either PS or medical devices. RESULTS: Thirty-four patients with incurable cancer were identified. MPS based on RC or SC was detected in 10 (29%) and 20 (59%) patients, respectively. Twenty-two of 34 patients who complained of pain, 10 (45%) had MPS based on RC and 20 (90%) had MPS based on SC. Age and central venous port were risk factors for MPS by multivariate analysis. CONCLUSION: A very high prevalence of MPS was detected in our study population. MPS should be considered when patients with incurable cancer complain of pain.

PRDM14 directly interacts with heat shock proteins HSP90α and glucose-regulated protein 78.

PRDM14 is overexpressed in various cancers and can regulate cancer phenotype under certain conditions. Inhibiting PRDM14 expression in breast and pancreatic cancers has been reported to reduce cancer stem-like phenotypes, which are associated with aggressive tumor properties. Therefore, PRDM14 is considered a promising target for cancer therapy. To develop a pharmaceutical treatment, the mechanism and interacting partners of PRDM14 need to be clarified. Here, we identified the proteins interacting with PRDM14 in triple-negative breast cancer (TNBC) cells, which do not express the three most common types of receptor (estrogen receptors, progesterone receptors, and HER2). We obtained 13 candidates that were pulled down with PRDM14 in TNBC HCC1937 cells and identified them by mass spectrometry. Two candidates-glucose-regulated protein 78 (GRP78) and heat shock protein 90-α (HSP90α)-were confirmed in immunoprecipitation assay in two TNBC cell lines (HCC1937 and MDA-MB231). Surface plasmon resonance analysis using GST-PRDM14 showed that these two proteins directly interacted with PRDM14 and that the interactions required the C-terminal region of PRDM14, which includes zinc finger motifs. We also confirmed the interactions in living cells by NanoLuc luciferase-based bioluminescence resonance energy transfer (NanoBRET) assay. Moreover, HSP90 inhibitors (17DMAG and HSP990) significantly decreased breast cancer stem-like CD24-  CD44+ and side population (SP) cells in HCC1937 cells, but not in PRDM14 knockdown HCC1937 cells. The combination of the GRP78 inhibitor HA15 and PRDM14 knockdown significantly decreased cell proliferation and SP cell number in HCC1937 cells. These results suggest that HSP90α and GRP78 interact with PRDM14 and participate in cancer regulation.

Establishment of a refined culture method for rat colon organoids.

BACKGROUND: Methods for the artificial three-dimensional (3D) culture of mouse and human small-intestinal and large-intestinal stem cells have been established with CD24+ or Paneth cell niches. In contrast, no studies have established stable 3D culture for rat colon stem cells. In this study, we established an advanced method for efficient rat colonic stem cell culture. METHODS: Using various tissue homogenates, we investigated the colonic organoid forming capacity under the TMDU protocol immediately adjacent to Ootani's 3D culture assembly in the same culture dish. Next, we examined whether the supernatant from the colon could be replaced by a colon homogenate. Finally, we identified the bioactive substances that were indispensable for efficient organoid culture using protein purification by three-step column chromatography and proteomic analysis with a quantitative nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: By combining Ootani's method with the TMDU protocol, we established a refined culture method for Lewis rat colon organoids, which we refer to as the modified TMDU protocol. Furthermore, we confirmed that PGE2 and galection-4 promoted rat colonic organoid formation. CONCLUSIONS: We established efficient rat colonic stem cell cultures in vitro. This success will contribute to the study of rat intestinal-disease models.

Inhibition of PRDM14 expression in pancreatic cancer suppresses cancer stem-like properties and liver metastasis in mice.

Pancreatic cancer is one of the most lethal types of cancer, with aggressive properties characterized by metastasis, recurrence and drug resistance. Cancer stem cells are considered to be responsible for these properties. PRDM14, a transcriptional regulator that maintains pluripotency in embryonic stem cells, is overexpressed in some cancers. Here, we assessed PRDM14 expression and the effects of PRDM14 knockdown on cancer stem-like phenotypes in pancreatic cancer. We observed that PRDM14 protein was overexpressed in pancreatic cancer tissues compared with normal pancreatic tissues. Using lentiviral shRNA-transduced pancreatic cancer cells, we found that PRDM14 knockdown decreased sphere formation, number of side population and cell surface marker-positive cells and subcutaneous xenograft tumors and liver metastasis in mice. This was accompanied by upregulation of some microRNAs (miRNAs), including miR-125a-3p. miR-125a-3p, a tumor suppressor that is down-regulated in pancreatic cancer, has been suggested to regulate the expression of the Src-family kinase, Fyn. In PRDM14-knockdown cells, Fyn was expressed at lower levels and downstream proteins were less activated. These changes were considered to cause suppression of the above cancer phenotypes. In addition, we used small interfering RNA (siRNA)-based therapy targeting PRDM14 in a mouse model of liver metastasis induced using MIA-PaCa2 cells, and this treatment significantly decreased metastasis and in vitro migration. Taken together, these results suggest that targeting the overexpression of PRDM14 suppresses cancer stem-like phenotypes, including liver metastasis, via miRNA regulation and siRNA-based therapy targeting it shows promise as a treatment for patients with pancreatic cancer.