Chin-Hsuan Hsieh

Total HLA class I loss in a sarcomatoid renal carcinoma cell line caused by the coexistence of distinct mutations in the two encoding β2-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-γ. β2-microglobulin (β2m) was not detected in RCC52 cells. Surprisingly, RCC52 cells harbor two mutations in the β2m 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 β2m 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 β2m 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 β2m 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.

Impetigo Herpetiformis with Gestational Hypertension: A Case Report and Literature Review

Background: Impetigo herpetiformis (IH) is a rare skin disorder that occurs during pregnancy. It was previously associated with high maternal and fetal mortality and morbidity, but now has a better prognosis. Case Report: We report a case of a pregnant woman with IH who presented with generalized erythematous pustular eruptions in the 32nd week of gestation. The IH progressed rapidly, and gestational hypertension was observed in the 36th week. The lesions did not subside, despite treatment with corticosteroids and phototherapy. She delivered a healthy male baby via cesarean section in the 37th week. One month after her delivery, her skin returned to normal, except for residual pigmentation, with complete recovery 3 months postpartum. Conclusion: An experienced medical team comprising obstetricians, dermatologists, perinatologists and neonatologists is critical to aggressively treat this life-threatening specific dermatosis of pregnancy and to prevent ensuing complications, such as fluid and electrolyte imbalance, secondary infection and placental insufficiency.

Withaferin A targeting both cancer stem cells and metastatic cancer stem cells in the UP-LN1 carcinoma cell model

Withaferin A targeting both cancer stem cells and metastatic cancer stem cells in the UP-LN1 carcinoma cell model Lai-Lei Ting1, Andy Shau-Bin Chou2, Chin-Hsuan Hsieh3, Shih-Chieh Hsiung3, See-Tong Pang3, Shuen-Kuei Liao4,5 1Department of Radiation Oncology, Cancer Center, Taipei Medical University Hospital, Taipei 110, Taiwan, China. 2Department of Radiology, Tzu-Chi General Hospital, Hualien 970, Taiwan, China. 3Department of Surgery, Division of Uro-Oncology, Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan, China. 4The PhD Program of Cancer Biology and Drug Discovery, Taipei Medical University, Taipei 110, Taiwan, China. 5Department of Research and Development, Vectorite Biomedica Inc., New Taipei City 221, Taiwan, China. Correspondence to: Dr. Shuen-Kuei Liao, The PhD Program of Cancer Biology and Drug Discovery, Taipei Medical University, Taipei 110, Taiwan, China. E-mail: liaosk@h.tmu.edu.tw Aim: As our understanding of cancer stem cell (CSC) biology improves, search for inhibitory agents of CSCs and metastatic CSCs (mCSCs) positive for CXCR4 is warranted. Withaferin A (WA), a withanolide extracted from the medicinal plant Withania somnifera, has been shown to exhibit anti-cancer effects through multiple mechanisms. Whether WA could selectively target CSCs, mCSCs, or nonCSCs of a gastrointestinal (GI) carcinoma tumor remains unclear. Methods: Side-population (SP) analysis, flow cytometric phenotyping and sorting, non-invasive imaging in conjunction with xenotransplantation, and immunohistology were used in this investigation. Results: Using the lymph node metastatic GI cancer cell line UP-LN1, consisting of CD44high/CD24low floating (F) and CD44low/CD24high adherent (A) cell subsets, this study demonstrated that as compared with parental UP-LN1 cells or A cells, WA preferentially reduced F-cell proliferation, tumor sphere formation, and SP cells in vitro in greater effi ciencies by apoptosis. This action was mechanistically mediated via the down-regulation of CXCR4/CXCL12 and STAT3/interleukin-6 axes, both of which are instrumental in the acquisition of metastatic ability. Attenuation of interferon-γ-induced CXCR4 expression in F cells by knockdown with siRNA or blocking with an anti-CXCR4 antibody, followed by Western blot analysis, showed signifi cantly reduced metastatic potential in vitro. The extent of in vitro anti-invasive effect of WA on the IFN-γ-treated F cells was signifi cantly greater than on the F cells without WA treatment, or F cells treated with control siRNA or with control IgG antibody. The observed in vitro effects of WA on the CSC and mCSC targeting were validated by data obtained with non-invasive imaging in NOD/SCID mouse xenotransplantation. Conclusion: WA could effi ciently block the formation of both CSCs and mCSCs in the UP-LN1 cell line, suggesting that WA may be considered an effective therapeutic agent for this type of GI malignancies.

Abstract 3622: Total loss of HLA class I expression by two sarcomatoid hepatocellular carcinoma cell lines sHCC29 and sHCC63 was caused by a ∼49-kbp deletion at chromosome 15q15 across the β2m gene locus

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Scanty information is available concerning the abnormalities in HLA class I antigen presentation in sarcomatoid hepatocellular carcinoma (sHCC), although this information can improve our understanding of sHCC-host interactions and assist in a rational design of T cell immunotherapy. In this study, we have established three sarcomatoid HCC cell lines, namely sHCC29, sHCC63, and sHCC74, from the tumor lesions in three patients; two of them were HBV carriers from which sHCC29 and sHCC63 were derived, while the other was a HCV carrier from which sHCC74 was generated. No viral DNA was detected in the culture supernatants or cell extracts from any of the three HCC cell lines by COBAS AmpliPre/TaqMan qPCR. In monolayer cell culture, both sHCC29 and sHCC74 showed similar elongated fibroblastic morphology, while sHCC63 exhibited a short-spindle shape and expanded in a density-sensitive manner. HLA class I molecules were abundantly expressed on sHCC74, but were not detected on sHCC29 and sHCC63 by cytofluorometric analysis. Cytoplasmic β2-microglobulin (β2m) was also undetected in the latter two cell lines, in spite of their baseline level of low-moderate cytoplasmic HLA class I heavy chain expression, which was 33-50% of that expressed by sHCC74 in terms of % positive cells. The observed HLA class I-loss phenotype was IFN-γ (300 μg/ml, 48 h)-uncorrectable. Inactivation of the β2m genes in both sHCC29 and sHCC63 was supported by the failure to detect their β2m mRNA transcripts and by the identification of a large (∼49,187 bp) deletion across the β2m gene locus at chromosome 15q15. This deletion, the largest reported to date, explains the loss of β2m protein and lack of assembly of HLA class I molecules in the sHCC29 and sHCC63 cell lines. Mapping of the deletion breaking point and validation by immunohistochemistry of β2m loss in the corresponding tumor lesions from which sHCC29 and sHCC63 were derived are currently underway. Citation Format: Wei-Yi Lei, Chin-Hsuan Hsieh, Chien-Chung Chang, Shao-Hsuan Wen, Chi-Tan Hu, Shuen-Kuei Liao. Total loss of HLA class I expression by two sarcomatoid hepatocellular carcinoma cell lines sHCC29 and sHCC63 was caused by a ∼49-kbp deletion at chromosome 15q15 across the β2m gene locus. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3622. doi:10.1158/1538-7445.AM2014-3622

Abstract 1198: Co-existence of epithelioid and fibroblastoid subsets in a sarcomatoid renal cell carcinoma: Comparative studies between clonal sublines and cytofluorometrically sorted cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Currently, understanding of the pathobiology of sarcomatoid renal cell carcinoma (sRCC) is incomplete. We previously reported the co-existence of epithelioid and fibroblastoid subsets in the sRCC cell line known as RCC52 with total loss of HLA class I expression through genetic and clonal cell studies (Cancer Immunol Immunother, 2009;58:395-408; Anticancer Res, 2013;35:4875-90). We argued that the conclusions drawn from data generated by clonal studies might have not been objective, since only a limited number of sublines from each morphologically distinct cell type were investigated. In this study, using anti-CD44 and anti-CD24 mAbs, we have been able to sort out the two subsets revealing that the CD44+/CD24- phenotype is for the epithelioid subset and the CD44+/CD24+ phenotype is for the fibroblastoid subset. The differential expression of two markers could also be used to identify the two morphologically distinct clonal sublines and the phenotypic results turned out to be consistent with those of sorted cell subsets, for examples: (i) in both differently prepared subsets, the respective phenotypic features in terms of the differential expression of CD44 and CD24 remained persistently stable after 12 in vitro passages; (ii) only epithelioid but not fibroblastoid cells expressed surface E-Cadherin; (iii) expression of surface CD105 was found much more abundant in epithelioid cells as compared with fibroblastoid ones; (iv) epithelioid cells exhibited stronger clonogenicity in semisolid agar as compared with fibroblastoid ones; and (v) fibroblastoid cells displayed greater migratory/invasive ability than epithelioid ones in in vitro assays. However, one inconsistent result between clonal and sorted cells was found that in xenotransplantation experiments with NOD/SCID immunodeficient mice, only epithelioid, but not fibroblastoid clonal sublines, developed tumors at the injection site, in contrast to sorted fibroblastoid cells which gave rise to larger tumor masses and faster growth rates than sorted epithelioid cells. The results obtained with sorted cells were more reflective of the whole tumor population and in vivo situations. Our findings highlight the difference in the functional properties of the two distinct subsets in this sRCC cell line. Further investigations with additional sRCC cell lines/tumors are needed to determine if the co-existence of epithelioid and fibroblastoid subsets, the total loss of HLA class I and other noted characteristics are indeed the common features of all sRCCs. Improved understanding of tumor heterogeneity, metastatic niche and stromal progression may have a profound consequence on disease progression and metastasis, and promises to open up new strategies for therapy of this type of malignancy. Citation Format: Chin-Hsuan Hsieh, Ming-Ling Kuo, Cheng-Keng Chuang, Shuen-Kuei Liao. Co-existence of epithelioid and fibroblastoid subsets in a sarcomatoid renal cell carcinoma: Comparative studies between clonal sublines and cytofluorometrically sorted cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1198. doi:10.1158/1538-7445.AM2014-1198

Abstract 3780: Tumorigenicity and partial clear cell conversion of the epithelioid, but not fibroblastoid, subset of a sarcomatoid renal carcinoma cell line.

The biology of sarcomatoid renal cell carcinoma (RCC) and its conversion from and to the clear cell RCC cells are not fully understood. In this study, we analyzed the sarcomatoid RCC cell line, RCC52, derived from a clear cell RCC lesion with extensive sarcomatoid differentiation. When grown as monolayers, RCC52 cells consisted of mostly epithelioid cells and partly fibroblastoid cells, and were all PAX-2 negative, suggestive of in vitro growth of the sarcomatoid, but not clear cell, component. Immunohistology of xenografts resulted from subcutaneous injection of tumor cells revealed that only the parental line and all epithelioid sublines tested could develop solid tumors consisting of mostly sarcomatoid cells and PAX-2+ clear cells in areas. Our findings revealed (i) the coexistence of the two phenotypically distinct epithelioid and fibroblastoid subsets in this sarcomatoid RCC52 cell line, (ii) the dissociation of the abilities to display tumorigenicity and migratory/invasive potential by the epithelioid and fibroblastoid subsets, respectively, (iii) the capability of only epithelioid subset to undergo partial conversion to a clear cell type, and (iv) the identification of epithelioid subset as the niche for cancer stem cells within the RCC52 cell line. Importantly, using CD44 and CD24 markers in cytofluorometric analysis, we identified the phenotypes of the epithelioid and fibroblastoid subsets to be CD44+/CD24− and CD44+/CD24+, respectively. Collectively, our findings highlight the phenotypical and functional properties of the two distinct subsets in a sarcomatoid RCC. Thus, improved understanding of tumor heterogeneity, metastatic niche and stromal progression has profound consequence of metastatic disease, and promises to open up new strategies for therapy of cancer. Citation Format: Chin-Hsuan Hsieh, Hung-Chang Chen, Cheng-Keng Chuang, See-Tong Pang, Ying-Hsu Chang, Shuen-Kuei Liao. Tumorigenicity and partial clear cell conversion of the epithelioid, but not fibroblastoid, subset of a sarcomatoid renal carcinoma cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3780. doi:10.1158/1538-7445.AM2013-3780

Abstract 1358: Epithelioid and fibroblastic cell elements coexisting within the sarcomatoid renal carcinoma RCC52 cell line ensue divergent routes of tumor differentiation and progression

The immunobiology of sarcomatoid renal cell carcinoma and its transformation from and to the clear cell RCC cells are not fully understood. We analyzed a human sarcomatoid RCC cell line known as RCC52, derived from a primary lesion of clear cell RCC with extensive sarcomatoid differentiation. When grown as monolayers, RCC52 cells were all PAX-2 negative and consisted of mostly epithelioid cells and occasionally spindle/fibroblastic cells, suggestive of in vitro growth of the sarcomatoid, but not clear cell, component of the tumor. PAX-2 is a transcription factor found in all major RCC types including clear cell RCCs, but not in sarcomatoid RCCs. We next isolated and maintained a panel of epithelioid and fibroblastic clonal sublines. As determined by cDNA microarray analysis, cancer stem cell (CSC) marker genes, such as ALDH1L1, PSCA and ABCA2, were expressed greater in the epithelioid sublines, while mesenchymal-like cell marker genes including vimentin, snail, fibronectin and MMPs were expressed in much higher levels in the fibroblastic counterpart. Xenotransplantation in NOD/SCID mice showed that in contrast to fibroblastic sublines failing to developed tumors, epithelioid sublines invariably developed tumors at the injection sites. These results point to the existence of CSCs in the epithelioid, but not fibroblastic sublines. In order to investigate the self-renewal/colongenicity ability of these two types of clonal sublines, we used soft agar assay to assess their differential anchorage-independent colony forming abilities. Results showed the mean colony number in the parental RCC52 cells at day 28 was 293.3 ± 81.3 colonies/well, in the epithelioid sublines, the mean colony number was 322.2 ± 92.0 colonies/well. In contrast, no visible colonies were detected in any of the fibroblastic sublines tested. These results are consistent with those obtained in the xenotransplantation studies. Another important observation was that only epithelioid sublines developed xenografts in which clear cell component which was identified by its positive PAX-2 reactivity by immunostaining in some areas of tissue sections. Surprisingly, xenografts resulting from subcutaneous injection of RCC52 cells and the original patient tumor shared similar histopathology in that mostly sarcomatoid cells were noted with occasional clear cell areas. In conclusion, our results strongly point to the capability of sarcomatoid RCCs to undergo dedifferentiation or transdifferentiation. Further investigations into the molecular events responsible for sarcomatoid transformation from clear cell RCCs and for the progression of clear cell RCCs to sarcomatoid ones using additional tissues and cell lines of either types are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1358. doi:1538-7445.AM2012-1358

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.

Abstract 4265: Identification of depressed HLA class I expression and CD44bright/CD24dim as the phenotype of floating but not adherent subpopulation harboring most tumor-initiating cells in the UP-LN1 carcinoma cell line

UP-LN1 is a poorly differentiated carcinoma cell line established from a lymph node (LN) metastatic lesion of the neck of a male patient with unknown primary. The CK7 − /CK20 + /CEA + /SCCA − phenotype detected in the original metastatic lesion and cultured cells led us to believe this cell line to be originated from a primary cancer of the gastrointestinal tract. UP-LN1 exhibited unique in vitro growth characteristics with naturally occurring floating (F) and adherent (A) cells. We hypothesize that the tumor-initiating cells (TICs) with cancer stem cell (CSC) properties may have differentially distributed in F and A cells of the UP-LN1 cell line contributing to cancer metastasis. We first made comparisons of TIC properties between F and A cells in terms of tumorigenicity in NOD/SCID mice. F and A cells were further examined for the expression of selected tumor markers and genes associated with TICs by DNA-microarray, RT-PCR and cytofluorometric analyses. Comparative sensitivities of the two cell types to cytolysis of non-MHC-restricted effector cells were also determined using 51 Cr release cytotoxicity assays. Results can be summarized as follows. (i) Injection of F cells at as low as 10 3 cells could initiate tumor formation in NOD/SCID mice, while an equivalent value for A cells was 10 5 . (ii) A panel of drug resistant gene mRNAs were tested, of which ABCG2, ABCA7, ABCB1, ABCC1 and ABCC4 were expressed in greater amounts in F cells as compared with A cells. (iii) F and A cells exhibited different phenotypes, CD44 bright /CD24 dim and CD44 dim /CD24 bright , respectively. (iv) Surface HLA class I expression was down-regulated in F cells, which could be restored after exposure to IFN-γ as low as 1 U/ml for 48 h. (v) F cells but not A cells exhibited extreme resistance to activated NK cells (CD56 dim /CD16 + cytotoxic subset) sorted out from peripheral blood mononuclear cells followed by IL-2 (100 U/ml, 72 h) activation. Moreover, F cells turned out to be highly sensitive to be modulated by IFN-γ to express CXCR4, a receptor of CXCL12. Up-regulation of surface CXCR4 expression with a concomitant down-regulation of cytoplasmic expression of CXCL12 was observed in F cells which are highly resistant to activated NK killing. Such modulation was found to be mediated via IFN-γ, a mediator which could be secreted by the immunomodulatory NK subset (CD56 bright /CD16 − ) within activated NK cell population. In summary, we have here identified resistance to activated NK killing, depressed HLA class I expression and CD44 bright /CD24 dim to be the phenotype of highly tumorigenic F cells, in which TICs/CSCs predominantly reside. IFN-γ-mediated induction of surface CXCR4 expression and enhancement of HLA class I expression in F cells might have occurred in the patient bearing UP-LN1 cells, resulting in invasive and metastatic events. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4265.

Abstract 3348: Immunophenotyping, xenotransplantability and molecular cytogenetic features of the sarcomatoid renal carcinoma cell line RCC52: Pathobiological significance

The immunobiology of sarcomatoid renal cell carcinoma (RCC) and how it has been transformed or progressed from clear cell-RCC are currently poorly understood. We analyzed a sarcomatoid renal carcinoma cell line (RCC52) derived from a primary clear cell renal carcinoma with intensive sarcomatoid differentiation, with respect to its immunophenotyping, molecular cytogenetic features and xenotransplantability. RCC52 cells grown as monolayers consisted of mostly small-sized epithelioid cells along with a small proportion of spindle/fibroblast-like cells, suggestive of growth of the sarcomatoid, but not clear cell component of the tumor. Cytofluorometric analysis revealed a phenotype of cell surface HLA-I−, E-CDH−, N-CDH−, EpCAM−, CD44+, CD54+, CD58+, URO1+ and URO10+ and cytoplasmic AE1+, E-CDH−, N-CDH+, Vimentin+, S100+ and VEGF+. Spectral karyotyping (SKY) show the chromosomal abnormalities in 22 metaphases examined to be 40-42 ,X,-Y[22]; der(2)t(2,3)(q35;q21)[20]; der(14)(14;17)(p11;q12)[22]; −3[11]; +7,+7[14]; −14[9]; −15[14]; −10[10]; −22[16]. Numerical imbalances were also assessed by comparative genomic hybridization, which are found to be consistent with the findings determined by banding and SKY. These results along with the documented genotype have established the identity of the RCC52 cell lines. Nude mouse xenografts resulting from RCC52 cell s.c. injection and the original tumor shared similar histopathology with mostly sarcomatoid elements with occasional clear cell areas, suggesting malignant potential of this cell line and the capability of sarcomatoid RCC cells to undergo transdifferentiation or dedifferentiation. The total HLA class I loss caused by the two distinctive mutations in the β2-microglobulin gene and loss of heterozygosity (LOH) in chromosome 15 observed recently (Cancer ImmunoI Immunother, 58:395-408, 2009) did not prevent sarcomatoid RCC52 cells from undergoing such transdifferentiation. Overall our results is of immunological (evasion of the host immunosurveillance) and pathobiological (progression/differentiation/transdifferentiation) significance, which forms the basis of further investigations with additional cell lines/ clones and tissues of clear cell-RCCs with sarcomatoid differentiation to confirm (i) loss HLA class I expression in most, if not all, sarcomatoid RCCs, and (ii) the proposed sequence of clear cell-RCC → fibroblast-like sarcomatoid RCC → epithelioid sarcomatoid RCC, which could in turn transdifferentiate into clear cell-RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3348.