Evaluation of anticancer agents using patient-derived tumor organoids characteristically similar to source tissues.

Patient-derived tumor xenograft models represent a promising preclinical cancer model that better replicates disease, compared with traditional cell culture; however, their use is low-throughput and costly. To overcome this limitation, patient-derived tumor organoids (PDOs) were established from human lung, ovarian and uterine tumor tissues, among others, to accurately and efficiently recapitulate the tissue architecture and function. PDOs were able to be cultured for >6 months, and formed cell clusters with similar morphologies to their source tumors. Comparative histological and comprehensive gene expression analyses proved that the characteristics of PDOs were similar to those of their source tumors, even following long-term expansion in culture. At present, 53 PDOs have been established by the Fukushima Translational Research Project, and were designated as Fukushima PDOs (F­PDOs). In addition, the in vivo tumorigenesis of certain F­PDOs was confirmed using a xenograft model. The present study represents a detailed analysis of three F­PDOs (termed REME9, 11 and 16) established from endometrial cancer tissues. These were used for cell growth inhibition experiments using anticancer agents. A suitable high-throughput assay system, with 96- or 384­well plates, was designed for each F­PDO, and the efficacy of the anticancer agents was subsequently evaluated. REME9 and 11 exhibited distinct responses and increased resistance to the drugs, as compared with conventional cancer cell lines (AN3 CA and RL95-2). REME9 and 11, which were established from tumors that originated in patients who did not respond to paclitaxel and carboplatin (the standard chemotherapy for endometrial cancer), exhibited high resistance (half-maximal inhibitory concentration >10 µM) to the two agents. Therefore, assay systems using F­PDOs may be utilized to evaluate anticancer agents using conditions that better reflect clinical conditions, compared with conventional methods using cancer cell lines, and to discover markers that identify the pharmacological effects of anticancer agents.

Family with sequence similarity 83, member B is a predictor of poor prognosis and a potential therapeutic target for lung adenocarcinoma expressing wild-type epidermal growth factor receptor.

Lung adenocarcinoma (ADC) patients with tumors that harbor no targetable driver gene mutation, such as epidermal growth factor receptor (EGFR) gene mutations, have unfavorable prognosis, and thus, novel therapeutic targets are required. Family with sequence similarity 83, member B (FAM83B) is a biomarker for squamous cell lung cancer. FAM83B has also recently been shown to serve an important role in the EGFR signaling pathway. In the present study, the molecular and clinical impact of FAM83B in lung ADC was investigated. Matched tumor and adjacent normal tissue samples were obtained from 216 patients who underwent complete lung resection for primary lung ADC and were examined for FAM83B expression using cDNA microarray analysis. The associations between FAM83B expression and clinicopathological parameters, including patient survival, were examined. FAM83B was highly expressed in tumors from males, smokers and in tumors with wild-type EGFR. Multivariate analyses further confirmed that wild-type EGFR tumors were significantly positively associated with FAM83B expression. In survival analysis, FAM83B expression was associated with poor outcomes in disease-free survival and overall survival, particularly when stratified against tumors with wild-type EGFR. Furthermore, FAM83B knockdown was performed to investigate its phenotypic effect on lung ADC cell lines. Gene silencing by FAM83B RNA interference induced growth suppression in the HLC-1 and H1975 lung ADC cell lines. FAM83B may be involved in lung ADC tumor proliferation and can be a predictor of poor survival. FAM83B is also a potential novel therapeutic target for ADC with wild-type EGFR.

TBX19 is overexpressed in colorectal cancer and associated with lymph node metastasis.

The T-box 19 (TBX19) gene encodes a transcription factor characterized by a highly conserved DNA-binding motif (T-box). Recent studies have revealed that TBX19 has been identified as one of the genes activated by KRAS mutations, and is upregulated in colon adenoma. These results indicate that TBX19 may work as an oncogene in colorectal cancer (CRC). However, the expression and role of TBX19 have yet to be investigated. Here, we investigated TBX19 mRNA and protein expressions in colon cancer cells or surgically resected CRC. We found that TBX19 mRNA expression was significantly increased in tumorous tissues compared to that in non-tumorous tissues, and increased TBX19 mRNA expression was associated with positive lymph node metastasis in our cohort. The expression of TBX19 mRNA was not correlated with that of TBX19 protein in tissue sample taken from the CRC patients. Moreover, TBX19 showed positive staining even in the normal colonic tissues and the adjacent non-tumorous tissues. These results suggest that the expression of TBX19 protein is not correlated with the expression of TBX19 mRNA. In addition, our results promote further investigations into the impact of TBX19 upregulation on colorectal carcinogenesis, as well as the underlying mechanisms.

Clinicopathological examination of dipeptidase 1 expression in colorectal cancer.

Dipeptidase 1 (DPEP1) is a zinc-dependent metalloproteinase that is fundamental in glutathione and leukotriene metabolism. DPEP1 was initially considered as a tumor suppressor gene in Wilms' tumor and breast cancer. However, it has been reported that DPEP1 is upregulated in colorectal cancers (CRCs) and high DPEP1 expression levels are associated with poorer patient survival. The role of DPEP1 genes in CRC, as well as their expression, requires investigation. Therefore, the present study investigated DPEP1 expression using reverse transcription-quantitative polymerase chain reaction or immunohistochemistry on surgically resected samples from CRC cases, and further examined the biological significance of DPEP1 by comparing the expression of the epithelial to mesenchymal transition (EMT) markers, including epithelial cadherin and Vimentin to clarify the function of DPEP1 in CRC, particularly in metastasis. The level of DPEP1 expression was identified to be significantly increased in tumorous tissue samples compared with that in non-tumorous tissue samples. In addition, increased DPEP1 mRNA expression levels were associated with positive lymph node metastasis in the included cohort. However, no positive correlations were observed between DPEP1 and EMT markers in the cohort. The results indiciates that further investigations into the upregulation of DPEP1 in colorectal carcinogenesis and the role of therapeutic or prognostic biomarkers are required.

Autocrine and Paracrine Interactions between Multiple Myeloma Cells and Bone Marrow Stromal Cells by Growth Arrest-specific Gene 6 Cross-talk with Interleukin-6.

The pathogenesis of multiple myeloma (MM) has not yet been fully elucidated. Our microarray analysis and immunohistochemistry revealed significant up-regulation of growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein with a structural homology with protein S, in bone marrow (BM) cells of MM patients. ELISA showed that the serum levels of soluble Gas6 were significantly increased in the MM patients when compared with healthy controls. Gas6 was overexpressed in the human CD138-positive MM cell line RPMI-8226. Exogenous Gas6 suppressed apoptosis induced by serum deprivation and enhanced cell proliferation of the MM cells. The conditional medium from the human BM stromal cell line HS-5 induced cell proliferation and anti-apoptosis of the MM cells with extracellular signal-regulated kinase, Akt, and nuclear factor-κB phosphorylation, which were reversed by the neutralizing antibody to Gas6 or IL-6. The TAM family receptor Mer, which has been identified as a Gas6 receptor, was overexpressed in BM cells of MM patients. The knockdown of Mer by siRNA inhibited cell proliferation, anti-apoptosis, and up-regulation of intercellular cell adhesion molecule-1 (ICAM-1) in MM cells stimulated by an HS-5 cell-conditioned medium. Furthermore, the Gas6-neutralizing antibody reduced the up-regulation of IL-6 and ICAM-1 induced by a HS-5 cell-conditioned medium in MM cells. The present study provides new evidence that autocrine and paracrine stimulation of Gas6 in concert with IL-6 contributes to the pathogenesis of MM, suggesting that Gas6-Mer-related signaling pathways may be a promising novel target for treating MM.

FAM83B is a novel biomarker for diagnosis and prognosis of lung squamous cell carcinoma.

Personalized therapy for non­small cell lung cancer (NSCLC), particularly lung adenocarcinoma, has recently been significantly improved by the discovery of various molecular targets. However, this has not been the case for lung squamous cell carcinoma (SCC). In the present study, we identified the family with sequence similarity 83, member B (FAM83B) as a candidate marker for SCC through a comprehensive gene expression analysis and examined its correlations with various clinicopathological factors. The subjects of this study consisted of 215 patients with NSCLC who underwent complete resection from 2005 to 2011 at the Fukushima Medical University Hospital (Fukushima, Japan). They included 102 patients with adenocarcinoma and 113 with SCC. FAM83B expression was first examined in some of the samples by gene expression analysis and western blotting, and then all clinical specimens were evaluated by immunohistochemistry (IHC). The relationship between the quantitative values for IHC and clinicopathological factors was statistically analyzed. The results showed that FAM83B mRNA expression was significantly higher in SCC than in normal lung or adenocarcinoma (P<0.0001). Immunoblot analysis also confirmed this trend. Specimens containing >10% positive area for FAM83B were judged as 'positive'; 94.3% (107/113) of SCC and 14.7% (15/102) of adenocarcinoma were positive. Patients were divided into two subgroups according to expression (54 high­expression and 53 low­expression patients); the high­expression group was associated with a better disease­free survival (DFS) rate (P=0.042, log­rank test). In conclusion, FAM83B may be a reliable diagnostic and prognostic biomarker for SCC. Detailed analyses of FAM83B function in lung cancer are required to understand how its expression is associated with better prognosis in SCC.

Expression screening of 17q12-21 amplicon reveals GRB7 as an ERBB2-dependent oncogene.

Gene amplification is a major genetic alteration in human cancers. Amplicons, amplified genomic regions, are believed to contain "driver" genes responsible for tumorigenesis. However, the significance of co-amplified genes has not been extensively studied. We have established an integrated analysis system of amplicons using retrovirus-mediated gene transfer coupled with a human full-length cDNA set. Applying this system to 17q12-21 amplicon observed in breast cancer, we identified GRB7 as a context-dependent oncogene, which modulates the ERBB2 signaling pathway through enhanced phosphorylation of ERBB2 and Akt. Our work provides an insight into the biological significance of gene amplification in human cancers.

Cathepsin L is highly expressed in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract that are diagnosed by c-kit staining in most cases. A lysosomal cysteine proteinase termed cathepsin L has been commonly associated with malignancy in several cancer types, but this finding has not been reported for GISTs. We analyzed the cathepsin L mRNA and protein expression in GISTs. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that cathepsin L levels were higher in GISTs than those in gastric or colorectal tumors; this finding was supported by results of the Western blot analysis. Immunohistochemistry revealed that cathepsin L was localized to the cytoplasm of GIST cells as an intense granular signal, which was not observed in the cells of leiomyoma, a mesenchymal tumor that was analyzed as a control specimen. Double immunofluorescence microscopy revealed that a portion of the granular signal colocalized with lysosome-associated membrane protein-1 (LAMP-1), which is a lysosomal marker. Moreover, immunohistochemical analysis of 43 tumor specimens revealed that 86.0% (n=37) were cathepsin-L positive, and this positivity was significantly correlated with c-kit positivity but not with other clinicopathological factors, including gender, age, region, size, mitosis and risk of recurrence. From these results, we conclude that cathepsin L is highly expressed in GISTs compared to its expression in other cancerous lesions; this identifies cathepsin-L as a new diagnostic marker for GISTs.

Constitutive activation of nuclear factor-kappaB is preferentially involved in the proliferation of basal-like subtype breast cancer cell lines.

Constitutive nuclear factor (NF)-kappaB activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF-kappaB activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF-kappaB activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF-kappaB activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal-like subtype, which is the most malignant form of breast cancer. Inhibition of constitutive NF-kappaB activation by expression of IkappaBalpha super-repressor reduced proliferation of the basal-like subtype cell lines. Expression levels of mRNA encoding NF-kappaB-inducing kinase (NIK) were elevated in several breast cancer cell lines, and RNA interference-mediated knockdown of NIK reduced NF-kappaB activation in a subset of the basal-like subtype cell lines with upregulated NIK expression. Taken together, these results suggest that constitutive NF-kappaB activation, partially dependent on NIK, is preferentially involved in proliferation of basal-like subtype breast cancer cells and may be a useful therapeutic target for this subtype of cancer.

NOTCH3 signaling pathway plays crucial roles in the proliferation of ErbB2-negative human breast cancer cells.

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by gamma-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (<1%). Taken together, these findings indicate that NOTCH3-mediated signaling rather than NOTCH1-mediated signaling plays an important role in the proliferation of ErbB2-negative breast tumor cells and that targeted suppression of this signaling pathway may be a promising strategy for the treatment of ErbB2-negative breast cancers.

FoxA1 as a lineage-specific oncogene in luminal type breast cancer.

The forkhead transcription factor FoxA1 is thought to be involved in mammary tumorigenesis. However, the precise role of FoxA1 in breast cancer development is controversial. We examined expression of FoxA1 in 35 human breast cancer cell lines and compared it with that of ErbB2, a marker of poor prognosis in breast cancer. We found that FoxA1 is expressed at high levels in all ErbB2-positive cell lines and a subset of ErbB2-negative cell lines. Down-regulation of FoxA1 by RNA interference significantly suppressed proliferation of ErbB2-negative and FoxA1-positive breast cancer cell lines. Down-regulation of FoxA1 also enhanced the toxic effect of Herceptin on ErbB2-positive cell lines through induction of apoptosis. Taken together with previous data that FoxA1 is a marker of luminal cells in mammary gland, our present results suggest that FoxA1 plays an important role as a lineage-specific oncogene in proliferation of cancer cells derived from mammary luminal cells.

Knock down of hSNF5/Ini1 causes cell cycle arrest and apoptosis in a p53-dependent manner.

hSNF5/Ini1 is a core component of the SWI/SNF complex and the gene is frequently mutated in aggressive pediatric rhabdoid tumors. Mechanisms of the malignant transformation, however, remain poorly understood. We analyzed HeLa cells treated with siRNA to the hSNF5/Ini1 mRNA. The resulting efficient and long-term suppression caused characteristic cell enlargement, cell cycle arrest in G1 phase, and subsequent modest apoptosis. Gene expression profiling of the hSNF5-down-regulated cells by cDNA microarray analysis revealed that a limited number of p53-responsive genes, especially p21, were up-regulated. The p53 protein level was also greatly enhanced, suggesting that loss of hSNF5/Ini1 induces a p53 signaling pathway irrelevant to the chk1/2 phosphorylation pathway. Some rhabdoid tumors with very low or no ARF expression were induced to undergo cell enlargement, growth arrest, and, in one case, apoptosis by ectopic expression of the p14ARF protein. These results may in part account for molecular mechanisms of rhabdoid tumor formation.

Novel clusters of highly expressed genes accompany genomic amplification in breast cancers.

Breast cancer is the most common cancer in women worldwide. To identify novel amplicons involved in the mammary carcinogenesis, we constructed gene expression maps of chromosomes in 35 human breast cancer cell lines and extracted six candidate amplicons containing highly expressed gene clusters on chromosomes 8, 17, and X. We also confirmed the presence of the identified amplicons in clinical specimens by Southern blot analysis. Highly expressed genes identified in the amplicons will contribute to the characterization of breast cancer phenotypes, thereby providing novel targets for anticancer therapies.

Differential responses of normal human coronary artery endothelial cells against multiple cytokines comparatively assessed by gene expression profiles.

Endothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1beta, tumor necrosis factor-alpha, interferon-beta, interferon-gamma, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions.

N-terminally truncated variant of the mouse GAIP/RGS19 lacks selectivity of full-length GAIP/RGS19 protein in regulating ORL1 receptor signaling.

The regulators of G protein signaling (RGS) are a family of proteins with conserved RGS domains and play essential roles in regulating G protein-mediated signal transduction and physiological events. GAIP/RGS19 (G alpha interacting protein, also classified as RGS19), a member of the RGS family, has been shown to negatively regulate the signaling of many G protein-coupled receptors, including the opioid receptors. Two GAIP/RGS19 mRNA variants, resulted from an alternative splicing of exon 2 of the GAIP/RGS19 gene, were identified in multiple mouse tissues. One of the transcripts consists of a complete set of exons and encodes a full-length GAIP/RGS19 protein, and the other does not have exon 2 and therefore encodes an N-terminal 22 residue truncated short GAIP/RGS19 protein. When co-expressed with either the opioid-receptor-like (ORL1) receptor or one of the mu, delta, and kappa opioid receptors, by transfecting dual-expression plasmids into COS-7 cells, the full-length GAIP/RGS19 was more effective than the N-terminally truncated variant and was more selective in regulating the ORL1 receptor signaling than in regulating the mu, delta, and kappa opioid receptors, as measured by the effectiveness to increase the agonist-stimulated GTPase activity and to reverse the agonist-induced inhibition of cyclic AMP accumulation. In the same assays, the N-terminally truncated GAIP/RGS19 did not distinguish ORL1 from the mu, delta, and kappa opioid receptors. In contrast, co-expression of RGS4 with either ORL1 or opioid receptors showed the selectivity of RGS4 for regulating opioid receptors was mu > kappa > delta > ORL1, an order completely different from that of GAIP/RGS19. The results suggest that GAIP/RGS19 prefers regulating ORL1 receptor signaling over other opioid receptors, and that the N-terminal domain of GAIP/RGS19 plays a crucial role in its receptor preference.

Transcriptional regulation of the human DNA methyltransferase 3A and 3B genes by Sp3 and Sp1 zinc finger proteins.

The DNMT3A (DNA methyltransferase 3A) and DNMT3B genes encode putative de novo methyltransferases and show complex transcriptional regulation in the presence of three and two different promoters respectively. All promoters of DNMT3A and DNMT3B lack typical TATA sequences adjacent to their transcription start sites and contain several Sp1-binding sites. The importance of these Sp1-binding sites was demonstrated by using a GC-rich DNA-binding protein inhibitor, mithramycin A, i.e. on the basis of decrease in the promoter activities and mRNA expression levels of DNMT3A and DNMT3B. Overexpression of Sp1 and Sp3 up-regulated the promoter activities of these two genes. The physical binding of Sp1 and Sp3 to DNMT3A and DNMT3B promoters was confirmed by a gel shift assay. Interestingly, Sp3 overexpression in HEK-293T cells (human embryonic kidney 293T cells) resulted in 3.3- and 4.0-fold increase in DNMT3A and DNMT3B mRNA expression levels respectively by quantitative reverse transcriptase-PCR, whereas Sp1 overexpression did not. Furthermore, an antisense oligonucleotide to Sp3 significantly decreased the mRNA levels of DNMT3A and DNMT3B. These results indicate the functional importance of Sp proteins, particularly Sp3, in the regulation of DNMT3A and DNMT3B gene expression.

A novel EID-1 family member, EID-2, associates with histone deacetylases and inhibits muscle differentiation.

An EID-1 (E1A-like inhibitor of differentiation-1) inhibits differentiation by blocking the histone acetyltransferase activity of p300. Here we report a novel inhibitor of differentiation exhibiting homology to EID-1, termed EID-2 (EID-1-like inhibitor of differentiation-2). EID-2 inhibited MyoD-dependent transcription and muscle differentiation. Unlike EID-1, EID-2 did not block p300 activity. Interestingly, EID-2 associated with class I histone deacetylases (HDACs). The N-terminal portion of EID-2 was required for the binding to HDACs. This region was also involved in the transcriptional repression and nuclear localization, suggesting the importance of the involvement of HDACs in the EID-2 function. These results indicate a new family of differentiation inhibitors, although there are several differences in the biochemical mechanisms between EID-2 and EID-1.