Nuclear Accumulation Of Beta-catenin Research Articles

Sema3C Signaling is an Alternative Activator of the Canonical WNT Pathway in Glioblastoma

Wnt signaling maintains normal and cancer stem cells. The Wnt pathway is frequently dysregulated in many cancers, underscoring it as a therapeutic target. Although Wnt inhibitors appear promising in many preclinical studies, they have failed uniformly in clinical trials. Molecular mechanisms of resistance are poorly defined. Further dissection of the precise mechanisms of Wnt pathway activation in specific tumor types is needed to develop new Wnt pathway inhibitors with less toxicity. Here, we identify an alternative activator of the Wnt pathway that may mediate resistance to upstream Wnt inhibition in glioblastoma. Glioma stem-like cells (GSCs) were enriched in defined media. GSCs were transduced with lentiviruses to knockdown or overexpress Sema3C or Wnt pathway components. Cell viability, proliferation, apoptosis, and self-renewal were assessed. Expression of Sema3C and Wnt pathway components were assessed in GSCs, mouse models of GBM, and human glioblastoma by qPCR, Western blot, and/or immunostaining. Beta-catenin subcellular localization was assessed by cell fractionation and immunofluorescence. GSC-derived orthotopic models of GBM were used to assess the impact of genetic or pharmacologic inhibition of Sema3C or Wnt pathway components alone or in combination on tumor growth and animal survival. The axonal guidance protein Sema3C promotes the tumorigenicity of GSCs through binding its NRP/PlxnD1 receptor complex leading to Rac1 activation. Sema3C signaling directs beta-catenin nuclear accumulation in a Rac1-dependent process, leading to transactivation of Wnt target genes. Sema3C-driven Wnt signaling occurred despite suppression of Wnt ligand secretion, suggesting that Sema3C may drive canonical Wnt signaling independent of Wnt ligand binding. In human glioblastoma, Sema3C expression and Wnt pathway activation were highly concordant. In a mouse model of glioblastoma, combined depletion of Sema3C and beta-catenin partner TCF1 extended animal survival more than single target inhibition alone. Sema3C signaling may represent an alternative mechanism of WNT pathway activation even when WNT ligand-receptor interaction is inhibited. Since Sema3C is overexpressed in >85% glioblastoma and is used to maintain GSCs but not normal neural progenitor cells, this pathway may represent a major mechanism of Wnt pathway activation and resistance to upstream Wnt pathway inhibitors in GSCs. Our data provide a therapeutic strategy to achieve clinically significant Wnt pathway inhibition in GSCs potentially without the toxicity of currently available WNT inhibitors.

CRIP1 suppresses BBOX1-mediated carnitine metabolism to promote stemness in hepatocellular carcinoma.

Carnitine metabolism is thought to be negatively correlated with the progression of hepatocellular carcinoma (HCC) and the specific molecular mechanism is yet to be fully elucidated. Here, we report that little characterized cysteine-rich protein 1 (CRIP1) is upregulated in HCC and associated with poor prognosis. Moreover, CRIP1 promoted HCC cancer stem-like properties by downregulating carnitine energy metabolism. Mechanistically, CRIP1 interacted with BBOX1 and the E3 ligase STUB1, promoting BBOX1 ubiquitination and proteasomal degradation, and leading to the downregulation of carnitine. BBOX1 ubiquitination at lysine 240 is required for CRIP1-mediated control of carnitine metabolism and cancer stem-like properties. Further, our data showed that acetylcarnitine downregulation in CRIP1-overexpressing cells decreased beta-catenin acetylation and promoted nuclear accumulation of beta-catenin, thus facilitating cancer stem-like properties. Clinically, patients with higher CRIP1 protein levels had lower BBOX1 levels but higher nuclear beta-catenin levels in HCC tissues. Together, our findings identify CRIP1 as novel upstream control factor for carnitine metabolism and cancer stem-like properties, suggesting targeting of the CRIP1/BBOX1/β-catenin axis as a promising strategy for HCC treatment.

Abstract LB111: Drug repurposing of niclosamide to regulate Wnt/beta-catenin signaling pathway through upregulating SFRP1 in colorectal cancer

Abstract Aim: This study investigated the inhibition of proliferation and invasiveness of colorectal cancer (CRC) by niclosamide via targeting the secreted Frizzled-related protein 1 (SFRP1) and the Wnt/beta-catenin signaling pathway. Background: CRC is the third most common cancer worldwide. Aberrant activation of various signaling pathways in CRC leads to its poor response to chemotherapy. Hyperactivation of Wnt/beta-catenin pathway is known to drive cell proliferation, invasion, and migration of CRC. Epigenetic silencing of extracellular Wnt inhibitors, such as secreted Frizzled-related proteins (SFRPs), has been shown to stabilize beta-catenin and subsequently activate the Wnt signaling pathway. Niclosamide is a clinically approved drug indicated for the treatment of tapeworm infections. We have recently reported the repurposing of niclosamide to potentiate chemotherapeutic drugs for treating CRC by STAT3 inhibition. Niclosamide was also reported to be a Wnt/beta-catenin inhibitor but the precise mechanism is not clear. Methods: The antiproliferative effect was assessed by MTT assay and colony formation assay. The mRNA level, protein expression, and methylation status of SFRP1, and sets of SFRP1 co-expressed genes in CRC were analyzed by the TCGA data portal. The mRNA expression of SFRP1 and CpG island methylator phenotype (CIMP) marker genes were measured by qRT-PCR. DNA methylation was analyzed by bisulfite genomic sequencing. Inhibition of Wnt/beta-catenin and its downstream targets were examined by Western blot analysis. Cell migration and invasion were investigated by wound healing and transwell chamber migration assay. The intracellular localization of beta-catenin was determined by immunofluorescence assay. Results: Niclosamide inhibited HCT116 cell proliferation and colony formation in a concentration-dependent manner. Compared with normal colon tissues, CRC expressed significantly lower level of SFRP1 mRNA. SFRP1 methylation was inversely correlated with its mRNA expression. The DNA-demethylating agent 5-azacytidine was shown to restore expression of SFRP1 and CIMP panel genes in HCT116 cells after 5-day treatment. However, SFRP1 mRNA level was only upregulated by 1-day niclosamide treatment, but not 3-day niclosamide treatment. The upregulation of SFRP1 by niclosamide was found not relevant to DNA demethylation according to bisulfite genomic sequencing data. Immunofluorescence and Western blotting analysis showed that the increased SFRP1 expression was accompanied by reduced expression and nuclear accumulation of beta-catenin. Moreover, a low concentration of niclosamide that has minimal effect on cell proliferation, was shown to significantly suppress migration and invasion of HCT116 cells. Conclusion: Niclosamide is a promising candidate for drug repurposing. It was shown to upregulate SFRP1 and subsequently suppress Wnt/beta-catenin signaling, thereby impairing CRC cell migration and invasion. Upregulation of SFRP1 by niclosamide was not dependent on DNA demethylation. Citation Format: Mia Mingxia Wu, Christy Wing Sum Tong, Vivi Wei Yan, Kenneth Kin-wah To. Drug repurposing of niclosamide to regulate Wnt/beta-catenin signaling pathway through upregulating SFRP1 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB111.

RARE-08. CYST FLUID CYTOKINES MAY PROMOTE EPITHELIAL-TO-MESENCHYMAL TRANSITION IN PEDIATRIC ADAMANTINOMATOUS CRANIOPHARYNGIOMA

BACKGROUNDDespite poor clinical outcomes, no targeted therapies have been established for the treatment of Adamantinomatous Craniopharyngioma (ACP). The only known genetic aberration is a mutation in CTNNB1 that results in the nuclear accumulation of beta-catenin. Nuclear beta-catenin is an established inducer of Epithelial-to-Mesenchymal Transition (EMT). ACP cyst fluid is enriched with pro-inflammatory and SASP cytokines, many of which are also directly implicated in EMT. We sought to investigate the role of EMT in ACP pathology.METHODSNormal human epithelial cells were cultured and treated with ACP cyst fluid (10%) for 1, 2, 4 and 8 days. Cell morphology was monitored by live cell brightfield microscopy. The expression of EMT associated genes, ZEB1, ZEB2, SNAI-1, SLUG, TWIST, E-Cadherin, Beta-Catenin and Vimentin was determined by RT-qPCR.RESULTSACP cyst fluid treated epithelial cells were markedly transformed into long, spindle-shaped cells. ACP cyst fluid treatment resulted in the progressive up-regulation of ZEB2 over 8 days (RQ=12.0; P<0.01), the progressive up-regulation of SNAI-1 over 4 days (RQ=5.1; P<0.05) and up-regulation of Vimentin (RQ=2.2; p<0.01), identified only on Day 8.CONCLUSIONACP cyst fluid can induce EMT-like changes in normal human epithelial cells. In conjunction with the frequency of beta-catenin mutation in ACP, it is possible that EMT plays a crucial role in the pathology of ACP. Understanding ACP pathology in the context of the EMT paradigm may aid the development of new targeted therapeutics.

Wnt pathway markers in molecular subgroups of glioblastoma

Glioblastoma (GBM) is a highly heterogeneous and aggressive brain tumor. Comprehensive genomic and transcriptomic analyses revealed that GBM segregates into three subgroups with characteristic signaling pathways. The Wnt pathway recently received increasing attention with the recognition of its importance in tumor development and recurrence through the promotion of glioma stem cells. As an extension of our previous translational studies, here we tested the possible interactions between key subgroup markers (IDH-1 R132H, EGFRvIII and both cytoplasmic and nuclear loss [c-/n-] of NF-1 expression) and the canonical (Wnt3a and beta-catenin) and non-canonical (Wnt5a and Fzd2) Wnt pathway markers by immunohistochemistry. These analyses revealed increased expression levels of both Wnt pathway markers with significant quantitative differences within, but not among subgroups. Wnt5a and Fzd2 levels were higher than the canonical marker levels in all subgroups. The strongest evidence for correlation between expression levels of the EGFRvIII subgroup marker and the Fzd2 Wnt marker was found, but weaker correlations also could be noted for IDH-1 R132H and NF-1 and some Wnt markers. The correlations detected between markers of the canonical and non-canonical pathways raised the possibility of cross-talk between the two pathways. Analyses of tumors with various NF-1 expression patterns (c+/n-; c-/n+ combined with c+/n+, and c-/n-) revealed that aberrant nuclear accumulation of NF-1 is accompanied by nuclear accumulation of beta-catenin, suggesting that they may act synergistically to define the tumor’s biology. Altogether, our study presents expression characteristics of Wnt ligands and receptors, and suggests complex molecular interactions in subgroups of GBM.

Shadow Cell Differentiation: A Comparative Analysis of Modes of Cell Death with Apoptosis and Epidermal/Trichilemmal Keratinization

Shadow cells are characterized by an eosinophilic cytoplasm and a ghost-like nuclear contour; the cell shape is preserved, in spite of nuclear disappearance. Shadow cell nests (SCNs) are frequently observed in pilomatricoma (PMX), where the transitional cells immediately adjacent to SCNs often have a crescent-shaped nucleus showing fragmentation similar to that of apoptotic bodies. They show nuclear accumulation of beta-catenin and DNA double strand breaks (as revealed by in situ 3′-tailing reaction or immunohistochemistry for single-stranded DNA [ssDNA]), while they are negative for cleaved caspase-3 or cleaved lamin A, suggesting that shadow cell differentiation (SCD) is a caspase-independent programmed cell death. SCD can be differentiated from epidermal keratinization (EK) and trichilemmal keratinization (TK) based on the expression pattern of beta-catenin, ssDNA, and caspase-14/CD138. SCD is observed not only in PMX, but also sometimes in basal cell carcinomas, gonadal teratomas, and various extra-cutaneous carcinomas. In particular, SCNs are found in 24% of endometrial adenoacanthoma and are derived from squamoid morules. This establishes a link between basaloid cells in PMX and squamoid morules in endometrial adenoacanthomas as common precursors of shadow cells. Overall, it is suggested that SCD is different from, but partly similar to, apoptosis and that SCD and EK/TK should be differentiated from the standpoint of cell death/differentiation.

LEF-1 is a Sensitive Marker of Cribriform Morular Variant of Papillary Thyroid Carcinoma.

Cribriform morular variant of PTC (CMV-PTC) frequently shows activation of the CTNNB1/Wnt pathway with nuclear accumulation of beta catenin. The utility of LEF-1, also in the CTNNB1/WNT pathway, in the diagnosis of CMV-PTC has not been previously studied. LEF-1 immunohistochemistry was performed on seven CMV-PTC, 52 benign cases and 101 malignant thyroid neoplasms. LEF-1 was scored by stain intensity (0 = no nuclear stain, 1 = weak nuclear stain, less than lymphocyte and 2 = strong nuclear stain, intense as lymphocyte) and percentage of positive cells at each intensity, for a maximum total score of 200. Sensitivity and specificity of LEF-1 stain for all cases and to differentiate between regular PTC and CMV-PTC was also calculated. Six of the seven CMV-PTCs showed ≥ 30% strong (2+) nuclear LEF-1 staining and a total score over 100. Beta catenin also showed strong and diffuse nuclear staining in these cases. One CMV-PTC was negative for both LEF-1 and beta catenin and did not have a history of FAP. All control PTC cases uniformly lacked LEF-1 staining at 2+ intensity. LEF-1 had a sensitivity of 86% and specificity of 98% for the diagnosis of CMV-PTC. LEF-1 is highly sensitive and specific marker for CMV-PTC, especially when used in the setting of a PTC neoplasm. The pattern of staining is important with ≥ 30% of cells showing strong 2+ nuclear staining having the highest combined sensitivity and specificity.

DOCK4 promotes loss of proliferation in glioblastoma progenitor cells through nuclear beta-catenin accumulation and subsequent miR-302-367 cluster expression.

Glioblastomas (GBM) are lethal primitive brain tumours characterized by a strong intra-tumour heterogeneity. We observed in GBM tissues the coexistence of functionally divergent micro-territories either enriched in more differentiated and non-mitotic cells or in mitotic undifferentiated OLIG2 positive cells while sharing similar genomic abnormalities. Understanding the formation of such functionally divergent micro-territories in glioblastomas (GBM) is essential to comprehend GBM biogenesis, plasticity and to develop therapies. Here we report an unexpected anti-proliferative role of beta-catenin in non-mitotic differentiated GBM cells. By cell type specific stimulation of miR-302, which directly represses cyclin D1 and stemness features, beta-catenin is capable to change its known proliferative function. Nuclear beta-catenin accumulation in non-mitotic cells is due to a feed forward mechanism between DOCK4 and beta-catenin, allowed by increased GSK3-beta activity. DOCK4 over expression suppresses selfrenewal and tumorigenicity of GBM stem-like cells. Accordingly in the frame of GBM median of survival, increased level of DOCK4 predicts improved patient survival.

Antisecretive and Antitumor Activity of Abiraterone Acetate in Human Adrenocortical Cancer: A Preclinical Study.

Patients with adrenocortical carcinoma (ACC) frequently suffer from cortisol excess, which portends a negative prognosis. Rapid control of cortisol hypersecretion and tumor growth are the main goals of ACC therapy. Abiraterone acetate (AA) is a potent inhibitor of 17alpha-hydroxylase/17,20-lyase, a key enzyme of adrenal steroidogenesis. This study sought to investigate the therapeutic use of AA in preclinical models of ACC. AA antisecretive and antiproliferative effects were investigated in vitro using NCI-H295R and SW13 ACC cell lines and human primary ACC cell cultures, as well as in vivo using immunodeficient mice. Steroid secretion, cell viability, and proliferation were analyzed in untreated and AA-treated ACC cells. The ability of AA to affect the Wnt/beta-catenin pathway in NCI-H295R cells was also analyzed. Progesterone receptor (PgR) gene was silenced by the RNA interference approach. The antitumor efficacy of AA was confirmed in vivo in NCI-H295R cells xenografted in immunodeficient mice. AA reduced the secretion of both cortisol and androgens, increased production of progesterone, and induced a concentration-dependent decrease of cell viability in the NCI-H295R cells and primary secreting ACC cultures. AA also reduced beta-catenin nuclear accumulation in NCI-H295R cells. AA administration to NCI-H295R-bearing mice enhanced progesterone levels and inhibited tumor growth. The cytotoxic effect of AA was prevented by either blocking PgR or by gene silencing. AA is able to inhibit hormone secretion and growth of ACC both in vitro and in vivo. It also reduces beta-catenin nuclear accumulation. The cytotoxic effect of AA seems to require PgR.

Gastric carcinoma with shadow cell differentiation in metastatic lymph nodes

A case of gastric carcinoma showing shadow cell differentiation (SCD) in metastatic regional lymph nodes was presented. The tumor histologically revealed features of poorly differentiated adenocarcinoma with focal squamoid components. The metastatic lymph node lesion exhibited similar histological features and was partially intermingled with shadow cell nests (SCNs) that were reminiscent of those seen in cutaneous pilomatricoma (PMX). Immunohistochemically, tumor cells around SCNs exhibited nuclear accumulation of beta-catenin and similar staining patterns for apoptosis-related molecules as those observed in PMX. This is the first report of gastric carcinoma with SCD and re-confirmed the common characteristics of carcinoma with SCD, such as modes of cell death and nuclear accumulation of beta-catenin.

Inhibition of the Tcf/beta-catenin complex increases apoptosis and impairs adrenocortical tumor cell proliferation and adrenal steroidogenesis.

To date, there is no effective therapy for patients with advanced/metastatic adrenocortical cancer (ACC). The activation of the Wnt/beta-catenin signaling is frequent in ACC and this pathway is a promising therapeutic target. To investigate the effects of the inhibition of the Wnt/beta-catenin in ACC cells. Adrenal (NCI-H295 and Y1) and non-adrenal (HeLa) cell lines were treated with PNU-74654 (5-200 μM) for 24-96 h to assess cell viability (MTS-based assay), apoptosis (Annexin V), expression/localization of beta-catenin (qPCR, immunofluorescence, immunocytochemistry and western blot), expression of beta-catenin target genes (qPCR and western blot), and adrenal steroidogenesis (radioimmunoassay, qPCR and western blot). In NCI-H295 cells, PNU-74654 significantly decreased cell proliferation 96 h after treatment, increased early and late apoptosis, decreased nuclear beta-catenin accumulation, impaired CTNNB1/beta-catenin expression and increased beta-catenin target genes 48 h after treatment. No effects were observed on HeLa cells. In NCI-H295 cells, PNU-74654 decreased cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. Additionally, in NCI-H295 cells, PNU-74654 decreased SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase 48 h after treatment. In Y1 cells, PNU-74654 impaired corticosterone secretion 24 h after treatment but did not decrease cell viability. Blocking the Tcf/beta-catenin complex inhibits the Wnt/beta-catenin signaling in adrenocortical tumor cells triggering increased apoptosis, decreased cell viability and impairment of adrenal steroidogenesis. These promising findings pave the way for further experiments inhibiting the Wnt/beta-catenin pathway in pre-clinical models of ACC. The inhibition of this pathway may become a promising adjuvant therapy for patients with ACC.

Overexpression of the Nek2 kinase in colorectal cancer correlates with beta-catenin relocalization and shortened cancer-specific survival.

The serine/threonine kinase Nek2 (NIMA-related kinase 2) regulates centrosome separation and mitotic progression, with overexpression causing induction of aneuploidy in vitro. Overexpression may also enable tumour progression through effects upon Akt signalling, cell adhesion markers and the Wnt pathway. The objective of this study was to examine Nek2 protein expression in colorectal cancer (CRC). Nek2 protein expression was examined in a panel of CRC cell lines using Western blotting and immunofluorescence microscopy. Nek2 and beta-catenin expression were examined by immunohistochemistry in a series of resected CRC, as well as their matched lymph node and liver metastases, and correlated with clinicopathological characteristics. Nek2 protein expression in all CRC lines examined was higher than in the immortalised colonocyte line HCEC. Nek2 overexpression was present in 86.4% of resected CRC and was significantly associated with advancing AJCC tumour stage and shortened cancer-specific survival. Elevated Nek2 expression was maintained within all matched metastases from overexpressing primary tumours. Nek2 overexpression was significantly associated with lower tumour membranous beta-catenin expression and higher cytoplasmic and nuclear beta-catenin accumulation. These data support a role for Nek2 in CRC progression and confirm potential for Nek2 inhibition as a therapeutic avenue in CRC.

Skin tumors with matrical differentiation: lessons from hair keratins, beta‐catenin and PHLDA‐1 expression

Pilomatricomas are tumors that emulate the differentiation of matrix cells of the hair follicle, showing cortical differentiation, with sequential expression of K35 and K31 keratins. Beta-catenin gene is frequently mutated in pilomatricoma, leading to beta-catenin nuclear accumulation, and to downstream expression of LEF1. Skin matrical tumors other than pilomatricoma are very rare, and comprise purely matrical tumors and focally matrical tumors. We aimed at studying cortical differentiation, beta-catenin pathway and expression of the follicular stem-cell marker PHLDA1 in a series of matrical tumors other than pilomatricoma. In 36 prospectively collected tumors, K31, K35, CK17, LEF1, HOXC13, beta-catenin and PHLDA1 expressions were evaluated. Five pilomatricomas were used as controls. In 18 purely matrical tumors (11 matrical carcinomas, 4 melanocytic matricomas, 3 matricomas) and 18 focally matrical tumors (11 basal cell carcinomas, 3 trichoepithelioma/trichoblastomas, 4 others), sequential K35, HOXC13 and K31 expressions were found, indicating cortical differentiation. Germinative matrix cells were always CK17-, and showed nuclear beta-catenin accumulation, with LEF1 and PHLDA1 expressions. Nuclear beta-catenin and LEF1 expression was highly conserved in matrical tumors, and suggested a common tumorigenesis driven by Wnt pathway activation. PHLDA1 was consistently expressed in matrical tumors and in areas of matrical differentiation.

Sa1993 Claudin-3 Expression Associates With Colonic Epithelial Differentiation and Loss of Its Expression Correlates With Colon Carcinogenesis and Poor Patient Survival

G A A b st ra ct s CDC5L expression was found in the nuclei of normal murine intestinal epithelial cells. Areas of the colonic epithelium in AhCre+Apcfl/fl and ApcMin/+ mice which showed evidence of Wnt signalling by nuclear localisation of beta catenin showed increased nuclear expression of SFRS2 and cytoplasmic localisation of CDC5L. CDC5L knockdown in HCT116 and HT29 cells resulted in significant inhibition of cell proliferation by both SRB and clonogenic assays (p,0.05). SFRS2 knockdown also resulted in increased nuclear expression of CDC5L in these cell lines. The malignant colonic lesions that developed in ApcMin/+Pten-/mice showed loss of nuclear beta catenin expression and reversal of the phenotype observed in ApcMin/+ mice, with nuclear CDC5L localisation once again. Conclusion Apc inactivation results in increased nuclear expression of SFRS2 in colonic epithelial cells in vivo. This coincides with translocation of CDC5L from the nucleus into the cytoplasm, which limits its ability to regulate the cell cycle and splice pre-mRNA. Acquisition of an additional mutation in Pten alters Wnt signalling further, causing reduced nuclear accumulation of beta catenin and allowing CDC5L to translocate back into the nucleus, where it possibly stabilises the tumor phenotype.

Abstract 3132: Beta-catenin regulates c-Myc and CDKN1A expression in breast cancer cells.

Abstract Basal-like breast cancer (BLBC) is associated with a poor prognosis and lacks therapeutic targets. The molecular pathways leading to the development of basal-like tumors are not well understood. We reported recently that nuclear and cytosolic accumulation of beta-catenin was enriched in BLBC, suggesting Wnt pathway activation in this specific subtype. c-Myc has been placed as a downstream effector of Wnt/beta-catenin pathway in colorectal cancer. However, exactly how the Wnt pathway regulates c-Myc in breast cancer and the biological significance of this regulation are poorly understood. In this study, we found that c-Myc is highly expressed in the basal-like subtype in comparison to the other four breast cancer subtypes by IHC staining. In addition, we confirmed the concordant expression of beta-catenin and c-Myc in basal-like breast tumors. Both c-Myc and beta-catenin proteins are highly expressed in the basal-like cell lines in comparison with non-basal-like cell lines. After silencing beta-catenin using siRNA, c-Myc expression was decreased in none-BLBC cells. In contrast, c-Myc mRNA and protein expression was up-regulated in the BLBC cell lines, suggesting that beta-catenin regulates c-Myc expression in a cell type dependent manner in breast cancer. Although we did not detect any TCF promoter activity in all breast cancer cell lines, decreased c-Myc promoter activity was observed after inhibiting beta-catenin by siRNA in non-BLBC cells; however, inhibition of beta-catenin or over-expression of dominant-negative LEF had no effect on c-Myc promoter activity in BLBC cell lines. In contrast, CDKN1A mRNA and p21 protein expression were significantly increased in all breast cancer cell lines upon beta-catenin silencing. Interestingly, inhibiting beta-catenin expression alone does not induce apoptosis in breast cancer cell lines despite c-Myc regulation,, but we observed a modest increase of cells in G1 and decrease of cells in S phase in the cell lines with beta-catenin silencing. Collectively, these findings suggest that the regulation of c-Myc in breast cancer cells is dependent on the molecular subtype, and that beta-catenin-mediated control of c-Myc and p21 may control the balance of cell death and proliferation in breast cancer by both TCF-dependent and TCF-independent mechanisms. Citation Format: Jinhua Xu, Yinghua Chen, Andrey Khramtsov, Dezheng Huo, Kathleen Goss, Olufunmilayo I. Olopade. Beta-catenin regulates c-Myc and CDKN1A expression in breast cancer cells. [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 3132. doi:10.1158/1538-7445.AM2013-3132

Abstract 4896: Real-time imaging of pancreatic cancer stem cells for identification of the selectively targeting therapy.

Abstract Identification and purification of cancer stem cells (CSCs) should offer a clue of the therapeutic targets, but their heterogeneous expansion remains the significant obstacle of studies. One of the solutions might be establishment of real-time monitoring of CSC, based on fluorescence imaging with the intrinsically low proteasome activity. The monitoring system in human pancreatic cancer cells proved directly the asymmetric division of CSC to generate non-CSC. Real-time chemoresistance of the CSCs and remarkable tumorigenicity (as few as 10 cells) indicated their preclinical significance. The synthetic lethal screening was applied to identify a novel compound targeting specifically the pancreatic CSCs. It inhibited nuclear accumulation of beta-catenin in the pancreatic CSCs. Anti-CSC effects of the compound were confirmed by in vitro dynamic images and in vivo tumor analyses. The real-time monitoring system of CSCs is an innovative strategy providing a visible target that should lead to the discovery of successful therapies directed toward CSCs of aggressive cancers. Citation Format: Shinji Tanaka, Rama Adikrisna, Satoshi Matsumura, Daisuke Ban, Takanori Ochiai, Takumi Irie, Atsushi Kudo, Noriaki Nakamura, Shoji Yamaoka, Shigeki Arii. Real-time imaging of pancreatic cancer stem cells for identification of the selectively targeting therapy. [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 4896. doi:10.1158/1538-7445.AM2013-4896 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Regulation of integrin αV subunit expression by sulfatide in hepatocellular carcinoma cells

Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.

Characterization of Wnt2 Overexpression in a Rat Granulosa Cell Line (DC3): Effects on CTNNB1 Activation1

WNTs comprise a family of secreted glycoproteins that are essential for normal embryonic development of the female reproductive system. The functional role that WNTs play in the postnatal ovary is poorly defined. We have shown previously that Wnt2 and Fzd4 mRNAs are expressed in granulosa cells of the postnatal rat ovary. Here we examine the effects of Wnt2 overexpression in a rat granulosa cell line (DC3) that displays characteristics of granulosa cells at an early stage of follicular development. We show that DC3 cells express a 7.7-kb Fzd4 mRNA transcript similar in size to that detected in the rat and human ovary. Our results demonstrate that Wnt2 overexpression in DC3 promotes cytosolic and nuclear accumulation of beta-catenin (CTNNB1), but does not stimulate CTNNB1/TCF-dependent (pGL3-OT) transcriptional activity. We show that chibby (CBY1), a nuclear CTNNB1-associated antagonist of the WNT pathway, is expressed in DC3 cells and associates with CTNNB1 in the presence and absence of Wnt2 overexpression, suggesting that Cby1 contributes to suppression of CTNNB1/TCF-dependent transcription in these cells. Our results show that Wnt2 overexpression in DC3 cells increases follistatin (Fst) mRNA expression and promotes resistance to activin-induced cell deletion. Taken together, our results suggest that WNT2 opposes activin activity in granulosa cells by up-regulating expression of the activin antagonist Fst in a CTNNB1/TCF-independent manner, and that rat granulosa cells express factors, including Cby1, that suppress CTNNB1/TCF-dependent signal transduction in the presence of a WNT signal.

Unusual Thyroid Tumors in a Young Woman

Question: A 24-year-old woman was referred to our hospital because of thyroid nodules. Physical examination revealed a painless nodule in the right lobe of the thyroid but no abnormal findings in the abdomen. The patient has no remarkable past history and laboratory studies, including blood count, chemistry, and thyroid function tests, were all within normal limits. Computed tomography revealed 2 thyroid tumors (Figure A) and fine needle aspiration from the right tumor suggested undifferentiated carcinoma. The patient underwent total thyroidectomy, which disclosed that both nodules were cancers with identical and unique histologic findings. They were composed of cribriform, morular, and papillary growth patterns (Figure B–D). Immunohistochemistry showed cytoplasmic and nuclear accumulation of beta-catenin (Figure E). The final diagnosis was cribriform-morular variant, a rare subtype of papillary thyroid carcinoma.View Large Image Figure ViewerDownload Hi-res image Download (PPT) What is a possible underlying gastrointestinal disease in this case? See the Gastroenterology web site (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and Images in GI. Patients with familial adenomatous polyposis (FAP) are at risk for extracolonic malignancies, including duodenal ampullary cancer, neurologic tumors, and papillary thyroid cancer. Approximately 1%–2% of FAP patients develop thyroid carcinoma, which is observed almost exclusively in young, female patients (male:female ratio, 1:20).1Groen E.J. Roos A. Muntinghe F.L. et al.Extra-intestinal manifestations of familial adenomatous polyposis.Ann Surg Oncol. 2008; 15: 2439-2450Crossref PubMed Scopus (174) Google Scholar The diagnosis of thyroid cancer precedes polyposis in one third of FAP cases.2Tomoda C. Miyauchi A. Uruno T. et al.Cribriform-morular variant of papillary thyroid carcinoma: clue to early detection of familial adenomatous polyposis-associated colon cancer.World J Surg. 2004; 28: 886-889Crossref PubMed Scopus (78) Google Scholar Thyroid cancer in FAP is often multiple and shows the unique histology known as cribriform-morular variant (CMV) of papillary carcinoma.2Tomoda C. Miyauchi A. Uruno T. et al.Cribriform-morular variant of papillary thyroid carcinoma: clue to early detection of familial adenomatous polyposis-associated colon cancer.World J Surg. 2004; 28: 886-889Crossref PubMed Scopus (78) Google Scholar CMV accounts for only 0.1%–0.2% of all papillary thyroid carcinoma, whereas >50% of CMV is associated with FAP.2Tomoda C. Miyauchi A. Uruno T. et al.Cribriform-morular variant of papillary thyroid carcinoma: clue to early detection of familial adenomatous polyposis-associated colon cancer.World J Surg. 2004; 28: 886-889Crossref PubMed Scopus (78) Google Scholar The histologic feature of CMV is a mixture of cribriform, morular, trabecular, and papillary growth pattern. Immunohistochemically, CMV invariably demonstrates aberrant nuclear and cytoplasmic accumulation of beta-catenin, which is not observed in other types of thyroid cancer.3Kurihara K. Shimizu S. Chong J.M. et al.Nuclear localization of immunoreactive β-catenin is specific to familial adenomatous polyposis in papillary thyroid carcinoma.Jpn J Cancer Res. 2000; 91: 1100-1102Crossref PubMed Scopus (34) Google Scholar In this case, the pathologic findings of thyroid cancer suggested underlying FAP, although the patient did not complain any polyposis-associated symptoms. Colonoscopy after thyroidectomy revealed hundreds of colorectal polyps (Figure F) without colorectal cancer. Polypectomy was performed for large polyps, all of which were low-grade tubular adenoma. The patient's father has also suffered from FAP and underwent total colectomy at the age of 35, although she did not know this until the diagnosis of her FAP. Germline mutation analysis by direct sequence revealed a nonsense mutation of APC exon 7.

Metadherin enhances the invasiveness of breast cancer cells by inducing epithelial to mesenchymal transition

The epithelial-mesenchymal transition (EMT) is a process in which polarized epithelial cells are converted into motile mesenchymal cells. During cancer development, EMT is conducive to tumor dissemination and metastatic spread. While overexpression of metadherin (MTDH) in breast cancer cell lines and tissues has been found to be associated with aggressive tumor behavior, its precise role in invasion and metastasis is largely unknown. Here we report that MTDH overexpression could significantly enhance the invasion and migration of breast cancer cells by inducing EMT. Metadherin overexpression led to upregulation of mesenchymal marker fibronectin, downregulation of epithelial marker E-cadherin, and the nuclear accumulation of beta-catenin. Also, transcription factors Snail and Slug were upregulated in breast cancer cells overexpressing MTDH. Overexpression of MTDH enhanced the invasiveness and migration ability of breast cancer cells in vitro. In addition, overexpression of MTDH led to increased acquisition of CD44(+) /CD24(-/low) markers that are characteristic of breast cancer stem cells. We also showed that NF-kappa was involved in the expression of EMT-related markers. Taken together, our results suggest that MTDH could promote EMT in breast cancer cells in driving the progression of their aggressive behavior.