Kruppel-like Factor 2 Inhibits Proliferation in Renal Angiomyolipoma via IL-6/JAK/STAT3 Signaling Pathway.

BACKGROUND/AIM: The transcription factor Kruppel-like factor 2 (KLF2) is thought to act as a tumor suppressor. However, its expression and function in renal angiomyolipomas (AMLs) remains unclear. This study aimed to investigate the expression and function of KLF2 in AML cells. MATERIALS AND METHODS: KLF2 was detected in AML tissues by immunohistochemistry and quantitative real-time polymerase chain reaction. The associations between KLF2 expression levels and clinicopathological features of patients with AMLs were analyzed. To explore its function in AMLs, KLF2 was over-expressed, and cell proliferation was assessed using cell counting kit-8 assay. Through Gene set enrichment analysis (GSEA) of RNA sequencing data, the signaling pathways regulated by KLF2 were predicted. The KLF2-regulated signaling pathway was validated by western blotting. RESULTS: KLF2 expression was dramatically suppressed in clinical samples of patients with AMLs. Low KLF2 expression was significantly associated with a larger tumor size and higher incidence of tumor hemorrhage (p=0.008 and p=0.009, respectively). In addition, KLF2 overexpression markedly inhibited SV7 and UMB cell survival and proliferation. GSEA and western blotting analysis revealed that KLF2 down-regulated the IL-6/JAK/STAT3 signaling pathway. CONCLUSION: Collectively, KLF2 mediated AML cell growth by regulating the IL-6/JAK/STAT3 signaling pathway. These results indicate that KLF2 plays an important role in AML progression and provide novel insights into diagnostic and therapeutic biomarkers for AMLs.

Gain of TPPP as a predictor of progression in patients with bladder cancer.

The present study investigated the role of tubulin polymerization promoting protein (TPPP) in the regulation of bladder cancer (BC) cell proliferation and migration, in addition to the association between TPPP gene copy number amplification and clinicopathological characteristics of BC. TPPP gene amplification was measured in human BC epithelial cells and samples obtained from 52 patients with BC via fluorescence in situ hybridization. TPPP gain was defined as mean TPPP copy number >2.2 per nucleus (cutoff). The neutrophil-to-lymphocyte ratio (NLR) was also obtained from the preoperative data of the patients. For in vitro assays, BC cell lines were transfected with either TPPP small interfering RNAs or scrambled control, following which cell proliferation and migration were determined using Cell Counting Kit-8 and Transwell migration assays, respectively. The percentage of cells with TPPP copy number amplification in the four BC epithelial cell lines (MGH-U1, -U1R, -U3, -U4) examined (86.0-100.0%) was found to be higher compared with that in the normal human uroepithelial cell lines (3.0 and 9.0%). Patients were divided into one- (1.9%), two- (55.8%), three- (7.7%), four- (26.9%) and five-copy (7.7%) types. Results calculated using Fisher's exact test indicated that the gain of TPPP in patients with BC associated significantly with age (P<0.05), advanced histological grade (P<0.001), tumor stage (P<0.05), histological type (P<0.001) and NLR (P<0.05). In MGH-U1R and MGH-U4 cells, cell proliferation and migration were revealed to be significantly lower following TPPP knockdown compared with those in cells transfected with the scrambled control. In conclusion, findings from the present study suggest that TPPP is important for cell proliferation, cell migration and BC progression, such that TPPP copy number assessment would be advised for preoperative urine cytology for urothelial neoplasia diagnosis.

Total HLA Class I Antigen Loss with the Downregulation of Antigen-Processing Machinery Components in Two Newly Established Sarcomatoid Hepatocellular Carcinoma Cell Lines.

Limited information is currently available concerning HLA class I antigen abnormalities in sarcomatoid hepatocellular carcinoma (sHCC). Here, we have analyzed the growth characteristics and HLA class I antigen status of four sHCC cell lines (sHCC29, sHCC63, sHCC74, and SAR-HCV); the first three were newly established in this study. Among the four, sHCC29 showed the highest growth rate in vitro and tumorigenicity in NOD-SCID mice. Unlike sHCC74 and SAR-HCV, both sHCC29 and sHCC63 had no detectable surface HLA class I antigen expression, alongside undetected intracellular ß 2-microglobulin (ß 2m) and marked HLA class I heavy chain and selective antigen-processing machinery (APM) component downregulation. The loss of ß 2m in sHCC29 and sHCC63 was caused by a >49 kb deletion across the B2M locus, while their downregulation of APM components was transcriptional, reversible by IFN-γ only in several components. ß 2m was also undetected in the primary HCC lesions of the patients involved, indicating its in vivo relevance. We report for the first time HLA class I antigen loss with underlying B2M gene deficiency and APM defects in 50% (2 of 4) of the sHCC cell lines tested. These findings may have implications for a proper design of T cell immunotherapy for the treatment of sHCC patients.

Differential expression of CD44 and CD24 markers discriminates the epitheliod from the fibroblastoid subset in a sarcomatoid renal carcinoma cell line: evidence suggesting the existence of cancer stem cells in both subsets as studied with sorted cells.

Epithelioid and fibroblastoid subsets coexist in the human sarcomatoid renal cell carcinoma (sRCC) cell line, RCC52, according to previous clonal studies. Herein, using monoclonal antibodies to CD44 and CD24 markers, we identified and isolated these two populations, and showed that CD44bright/CD24dim and CD44bright/CD24bright phenotypes correspond to epithelioid and fibroblastoid subsets, respectively. Both sorted subsets displayed different levels of tumorigenicity in xenotransplantation, indicating that each harbored its own cancer stem cells (CSCs). The CD44bright/CD24bright subset, associated with higher expression of MMP-7, -8 and TIMP-1 transcripts, showed greater migratory/invasive potential than the CD44bright/CD24dim subset, which was associated with higher expression of MMP-2, -9 and TIMP-2 transcripts. Both subsets differentially expressed stemness gene products c-Myc, Oct4A, Notch1, Notch2 and Notch3, and the RCC stem cell marker, CD105 in 4-5% of RCC52 cells. These results suggest the presence of CSCs in both sRCC subsets for the first time and should therefore be considered potential therapeutic targets for this aggressive malignancy.

Co-existence of epithelioid and fibroblastoid subsets in a sarcomatoid renal carcinoma cell line revealed by clonal studies.

BACKGROUND: The biology of sarcomatoid renal cell carcinoma (RCC) and its conversion from and to the clear cell RCC are not fully-understood. We aimed to analyze the sarcomatoid RCC cell line, RCC52, derived from a lymph node metastatic lesion consisting mostly of sarcomatoid RCC cells with occasional clear cell areas. MATERIALS AND METHODS: Representative clonal epithelioid and fibroblastoid sublines isolated from the RCC52 cell line were analyzed alongside the parental line. Cytofluorometric and western blot analyses were used for phenotypic study. Xenotransplantation and in vitro invasive assays were used to determine tumorigenicity and invasiveness. Immunohistology in conjunction with antibodies to paired box gene-2 (PAX2) were used to determine if xenografts or tumor biopsies had the clear cell component. RESULTS: RCC52 cells grown as monolayers in vitro were all PAX2-negative, and consisted mostly of epithelioid cells and partly of fibroblastoid cells as noted in a previous study, confirming the co-existence of these two cell types in the in vitro growth of exclusive sarcomatoid RCC cells. Immunohistology revealed that the parental line and all epithelioid sublines tested were able to develop into solid tumors consisting mostly of sarcomatoid cells with PAX2-positive clear cells in some areas. The RCC stem cell marker CD105 was selectively expressed by a small proportion of the epithelioid, but not fibroblastoid, sublines, which was in line with the tumorigenic property of the epithelioid sublines containing cancer stem cells (CSCs). In contrast, only fibroblastoid sublines exhibited migratory/invasive properties, as determined by in vitro assays. CONCLUSION: Our findings confirm the presence of two distinct subsets in the RCC52 line, and suggest the epithelioid subset being able to de-differentiate to clear cells, albeit partially, and harboring CSCs as an emerging therapeutic target in order to achieve effective treatment of this malignancy.

Tumor dormancy resulting from subcutaneous injection to SCID mice with cultured nasopharyngeal carcinoma cells is mediated via IFN-γ induction of a highly differentiated phenotype.

The mechanisms underlying tumor dormancy in human primary lesions and bone marrow metastases of nasopharyngeal carcinoma (NPC) are still not completely understood. The aim of this study was to determine differences in the fates of cultured primary NPC (P-NPC) cells, interferon-γ-transduced primary NPC (IFN-γ-P-NPC) cells, bone marrow metastatic NPC (BM-NPC), and IFN-γ-transduced BM-NPC (IFN-γ-BM-NPC) cells following xenotransplantation into these four groups of SCID mice through subcutaneous injection of 5×10(6) cells/site/animal (4 animals/group). The injected mice were monitored for tumor development at the sites of injection. In only the group injected with IFN-γ-P-NPC cells, the resulting nodules remained small throughout the 60-day observation period after injection, but gradually became palpably prickly. Histopathological examination revealed that these lesions invariably consisted of mostly structures of horny pearls and keratin bridges with occasional apoptotic and degenerative cells. In contrast, animals injected with nontransduced-P-NPC cells developed tumors progressively with occasional central necroses. In the two groups injected with IFN-γ-NPC-BM and NPC-BM cells, progressive growths of tumors were noted, with the latter being at slightly faster rates, whereas the xenografts of both groups showed a poorly differentiated phenotype with abundant vascularity. The study results highlight the high susceptibility of P-NPC but not BM-NPC following IFN-γ gene transfer to the induction of tumor dormancy, which is mediated via induced cell differentiation. Thus, induced cell differentiation could provide a new mechanism by which tumor dormancy is induced.

Induction of metastatic cancer stem cells from the NK/LAK-resistant floating, but not adherent, subset of the UP-LN1 carcinoma cell line by IFN-γ.

As an advanced status of cancer stem cells (CSCs), metastatic CSCs (mCSCs) have been proposed to be the essential seeds that initiate tumor metastasis. However, the biology of mCSCs is poorly understood. In this study, we used a lymph node (LN) metastatic CEA-producing carcinoma cell line, UP-LN1, characterized by the persistent appearance of adherent (A) and floating (F) cells in culture, to determine the distribution of CSCs and mechanisms for the induction of mCSCs. F and A cells displayed distinct phenotypes, CD44(high)/CD24(low) and CD44(low)/CD24(high), respectively. The CSC-rich nature of F cells was typified by stronger expression of multiple drug resistance genes and a 7.8-fold higher frequency of tumor-initiating cells in NOD/SCID mice when compared with A cells. F cells showed a greater depression in HLA class I expression and an extreme resistance to NK/LAK-mediated cytolysis. Moreover, the NK/LAK-resistant F cells were highly susceptible to IFN-γ-mediated induction of surface CXCR4, with concomitant downregulation of cytoplasmic CXCL12 expression, whereas these two parameters remained essentially unchanged in NK/LAK-sensitive A cells. Following the induction of surface CXCR4, enhanced migratory/invasive potential of F cells was demonstrated by in vitro assays. Confocal immunofluorescence microscopy showed the two distinct phenotypes of F and A cells could be correspondingly identified in monodispersed and compact tumor cell areas within the patient's LN tumor lesion. In response to IFN-γ or activated NK/LAK cells, the CXCR4(+) mCSCs could be only induced from the CSCs, which were harbored in the highly tumorigenic CD44(high)/CD24(low) F subset. Our results revealed the complexity and heterogeneity of the CSC of this cell line/tumor and the differential immunomodulatory roles of F and A cells. A better understanding of the interactions among different classes of CSCs and their niches may assist us in eradicating the CSCs/mCSCs through targeted immunotherapy, chemotherapy, or both.

Epstein-Barr virus-infected cell line TCC36B derived from B lymphocytes infiltrating renal pelvis urothelial carcinoma.

UNLABELLED: This study reports an initial analysis of an EBV-infected B cell line (TCC36B), established from an urothelial carcinoma (UC) lesion of the renal pelvis. MATERIALS AND METHODS: Cytofluorometric and G-banding analyses were performed for phenotyping and cytogenetics. PCR was used to detect EBV DNA, and sequence analysis to investigate mutations and deletions of the latent membrane protein (LMP)-1 gene of EBV. RESULTS: TCC36B cells proliferated in vitro and showed positivity for surface CD19, CD20, HLA-DR and IgG(λ), indicating that they belong to B-cells. Cytogenetic analysis showed 46,XX with a unique clonal abnormality of dup(2)(p13p25). EBV DNA was detected in TCC36B cells. Sequence analysis revealed a 30-bp deletion and 7 point mutations on the LMP-1 gene in TCC36B cells. CONCLUSION: These results suggest the involvement of an EBV variant in the pathogenesis of UC. This cell line should thus facilitate further investigations on the aetiological role of EBV in urothelial cancer.

Total HLA class I loss in a sarcomatoid renal carcinoma cell line caused by the coexistence of distinct mutations in the two encoding beta2-microglobulin genes.

In renal cell carcinoma (RCC), HLA class I downregulation has been found in about 40% of the lesions examined. Since only scanty information is available about the molecular basis of these defects, we have investigated the mechanism(s) underlying HLA class I antigen downregulation or loss in six RCC cell lines. Five of them express HLA class I antigens although at various levels; on the other hand, HLA class I antigens are not detectable on the remaining cell line, the RCC52 cell line, belonging to a sarcomatoid subtype, even following incubation with IFN-gamma. beta(2)-microglobulin (beta(2) m) was not detected in RCC52 cells. Surprisingly, RCC52 cells harbor two mutations in the beta ( 2 ) m genes in exon 1: a single G deletion (delG) in codon 6, which introduces a premature stop at codon 7, and a CT dinucleotide deletion (delCT), which leads to a premature stop at codon 55. Analysis of eight clonal sublines isolated from the RCC52 cell line showed that the two beta ( 2 ) m gene mutations are carried separately by RCC52 cell subpopulations. The delG/delCT double mutations were detected in two sublines with a fibroblast-like morphology, while the delCT mutation was detected in the remaining six sublines with an epithelial cell morphology. Furthermore, loss of heterozygosity (LOH) of the beta ( 2 ) m gene at STR D15S-209 was found only in the epithelioid subpopulation, indicating loss of one copy of chromosome 15. Immunostaining results of the tumor lesion from which the cell line RCC52 was originated were consistent with the phenotyping/molecular findings of the cultured cells. This is the first example of the coexistence of distinct beta ( 2 ) m defects in two different tumor subpopulations of a RCC, where loss of one copy of chromosome 15 occurs in one of the subpopulations with total HLA class I antigen loss.