The primary aim of this report was to examine the significance of increased DNA sequence copy number (gene amplification)
in human prostate cancers. Three methodologies (chromosome microdissection, ...comparative genomic hybridization, and fluorescence
in situ hybridization) were combined to (a) identify a common region of gene amplification in human prostate cells and (b)
evaluate in patient samples the prevalence of this genetic change in both primary and recurrent prostate samples. The results
of chromosome microdissection revealed a common amplified band region (8q24.1-24. 2) in two prostate cases with cytological
evidence of gene amplification (double minutes). Fluorescence in situ hybridization using the 8q microdissection probe was
performed on fresh tumor touch preparations from 44 randomly selected prostatectomy specimens. Amplification of DNA sequences
from 8q24 was observed in 4 (9%) of 44 cases. Four of the 44 patients in this series presented with a positive lymph node
at initial diagnosis and 3 of these 4 patients showed 8q amplification. Because of this finding, comparative genomic hybridization
and fluorescence in situ hybridization were performed on tumor cells from nine prostate cancer patients with recurrent disease.
In eight of nine cases a gain of DNA sequences encompassing 8q24 was observed. Taken together with other evidence implicating
8q gain in prostate cancer progression, these results suggest that the analysis of this genetic change may have diagnostic
utility as a marker of prostate cancer progression.
Hepatocellular carcinoma (HCC) is a highly vascularized tumor with poor clinical outcome. Our previous work has shown that eukaryotic initiation factor 5A2 (EIF5A2) over-expression enhances HCC cell ...metastasis. In this study, EIF5A2 was identified to be an independent risk factor for poor disease-specific survival among HCC patients. Both in vitro and in vivo assays indicated that ablation of endogenous EIF5A2 inhibited tumor angiogenesis by reducing matrix metalloproteinase 2 (MMP-2) expression. Given that MMP-2 degrades collagen IV, a main component of the vascular basement membrane (BM), we subsequently investigated the effect of EIF5A2 on tumor vasculature remodeling using complementary approaches, including fluorescent immunostaining, transmission electron microscopy, tumor perfusion assays and tumor hypoxia assays. Taken together, our results indicate that EIF5A2 silencing increases tumor vessel wall continuity, increases blood perfusion and improves tumor oxygenation. Additionally, we found that ablation of EIF5A2 enhanced the chemosensitivity of HCC cells to 5-Fluorouracil (5-FU). Finally, we demonstrated that EIF5A2 might exert these functions by enhancing MMP-2 activity via activation of p38 MAPK and JNK/c-Jun pathways.
This study highlights an important role of EIF5A2 in HCC tumor vessel remodeling and indicates that EIF5A2 represents a potential therapeutic target in the treatment of HCC.
Pirh2 is a Ring-H2 domain containing E3 ubiquitin ligase that targets several important tumor suppressor genes for proteasomal degradation. Overexpression of Pirh2 is frequently detected in many ...clinical tumor tissues including hepatocellular carcinoma (HCC). However, the molecular mechanism of Pirh2 activation in tumorigenesis still remains poorly understood. In this study, we find a Pirh2-binding protein, SCYL1 binding protein 1 (SCYL1BP1), that can promote the ubiquitin-dependent degradation of Pirh2. SCYL1BP1 colocalized with Pirh2 in the cytoplasm and prevented its localization to the nucleus. Ectopic expression of SCYL1BP1 increased the expression of p53 and further inhibited the G(1)/S transition of HCC cell lines. Conversely, knock down of SCYL1BP1 restored the expression of Pirh2 and inhibited p53 at protein level. Functional assays found that reintroduction of SCYL1BP1 into HCC cell lines significantly inhibited cell proliferation, foci formation, colony formation in soft agar and tumor formation in nude mice, suggesting the strong tumor-suppressive function of SCYL1BP1 in HCC progression. Furthermore, SCYL1BP1 was found to be frequently downregulated in HCC clinical specimens compared to their paired non-tumor tissues by immunohistochemical staining. Taken together, our data suggested that the interaction of SCYL1BP1/Pirh2 could accelerate Pirh2 degradation through an ubiquitin-dependent pathway. SCYL1BP1 may function as an important tumor suppressor gene in HCC development.
Abstract
Tumor relapse after therapy typifies hepatocellular carcinoma (HCC) and is believed to be attributable to residual cancer stem cells (CSCs) that survive initial treatment. We have previously ...identified a CSC population derived from HCC that is characterized by the expression of the transmembrane glycoprotein, CD133. Despite our growing knowledge of the importance of a functional CD133+ liver CSC subset in driving HCC, the regulatory mechanism of CD133 is not known. Epigenetic changes are believed to be essential in the control of cancer and stem cells. We report here the dynamic epigenetic regulation of the functional liver CSC marker CD133 by promoter methylation and miR-142-3p regulation. Unlike in other tumor types, we found DNA methylation to only play a minor role in the control of CD133 expression in HCC. More importantly, our results revealed that miR-142-3p plays an integral part in the direct targeting of CD133. The interaction between the 3’UTR of CD133 and miR-142-3p was identified by in silico prediction and substantiated by luciferase reporter analysis. Expression of CD133 was found to be inversely correlated with miR-142-3p in a panel of liver cell lines and HCC clinical samples. Functional studies with miR-142-3p stably transduced in HCC cells demonstrated a diminished ability to self-renew, initiate tumor growth, invade, migrate, induce capillary tube formation in endothelial cells and resist standard chemotherapy. Rescue experiments whereby CD133 and miR-142-3p is simultaneously overexpressed compensated the deregulated ability of the cells to confer these cancer and stem cell-like features. In summary, our findings suggestion promoter methylation to only play a minor role in the regulation of CD133 in HCC; and that miR-142-3p directly targets CD133 to regulate its ability to confer cancer and stem cell-like features in HCC.
Citation Format: Kai Yu Ng, Stella Chai, Man Tong, Pak Shing Kwan, Yuen Piu Chan, Terence Kin Wah Lee, Nathalie Wong, Xin-Yuan Guan, Stephanie Ma. Regulatory role of miR-142-3p on the functional hepatic cancer stem cell marker CD133. 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 LB-53. doi:10.1158/1538-7445.AM2014-LB-53
Amplification of 1q is one of the most frequent chromosomal alterations in human hepatocellular carcinoma (HCC). In this study we identified and characterized a novel oncogene, Maelstrom (MAEL), at ...1q24. Amplification and overexpression of MAEL was frequently detected in HCCs and significantly associated with HCC recurrence (P = 0.031) and poor outcome (P = 0.001). Functional study demonstrated that MAEL promoted cell growth, cell migration, and tumor formation in nude mice, all of which were effectively inhibited when MAEL was silenced with short hairpin RNA (shRNAs). Further study found that MAEL enhanced AKT activity with subsequent GSK-3beta phosphorylation and Snail stabilization, finally inducing epithelial-mesenchymal transition (EMT) and promoting tumor invasion and metastasis. In addition, MAEL up-regulated various stemness-related genes, multidrug resistance genes, and cancer stem cell (CSC) surface markers at the messenger RNA (mRNA) level. Functional study demonstrated that overexpression of MAEL increased self-renewal, chemoresistance, and tumor metastasis. Conclusion: MAEL is an oncogene that plays an important role in the development and progression of HCC by inducing EMT and enhancing the stemness of HCC. (Hepatology 2014;59:531-543) PUBLICATION ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common fatal malignancies but the molecular genetic basis of this disease remains unclear. By using genome-wide methylation profiling analysis, we ...identified CLDN3 as an epigenetically regulated gene in cancer. Here, we investigated its function and clinical relevance in human HCC. CLDN3 downregulation occurred in 87/114 (76.3%) of primary HCCs, where it was correlated significantly with shorter survival of HCC patients (P=0.021). Moreover, multivariate cyclooxygenase regression analysis showed that CLDN3 was an independent prognostic factor for overall survival (P=0.014). Absent expression of CLDN3 was also detected in 67% of HCC cell lines, which was significantly associated with its promoter hypermethylation. Ectopic expression of CLDN3 in HCC cells could inhibit cell motility, cell invasiveness, and tumor formation in nude mice. Mechanistic investigations suggested through downregulation of GSK3B, CTNNB1, SNAI2, and CDH2, CLDN3 could significantly suppress metastasis by inactivating the Wnt/β-catenin-epithelial mesenchymal transition (EMT) axis in HCC cells. Collectively, our findings demonstrated that CLDN3 is an epigenetically silenced metastasis suppressor gene in HCC. A better understanding of the molecular mechanism of CLDN3 in inhibiting liver cancer cell metastasis may lead to a more effective management of HCC patients with the inactivation of CLDN3.
Purpose: Many studies suggest that Her2/neu play an important role in neoadjuvant endocrine therapy. This study aimed to determine
whether the level of Her2/neu expression in advanced breast cancer ...changes after antiaromatase neoadjuvant treatment, as well
as to identify the relationship between Her2/neu expression and response to this kind of therapy.
Experimental Design: Thirty-six postmenopausal patients with hormonal receptor-positive primary breast cancer were included in a study of three
monthly cycles of neoadjuvant endocrine therapy with either Aromasin (25 mg daily) or Femara (2.5 mg daily). Immunohistochemistry
(IHC) and fluorescence in situ hybridization (FISH) for Her2/neu were conducted both on pretreatment biopsies and surgical tumors.
Results: Using IHC, 5 of 36 (13.9%) of the patients had a Her2/neu overexpression after treatment, as compared with 16 of 36 (44.4%)
before. Meanwhile, there was no change in 21 (58.3%) patients, and through FISH, there was a change from amplification to
no amplification in 15 (41.7%) patients. The response rate to the treatment was 75% for Her2/neu (+) tumors and 35% for Her2/neu
(−) tumors ( P = 0.017) while FISH was performed. The response rate was also significantly affected by the decrease in Her2/neu status after
the treatment, with 73% of the tumors showing decreased Her2/neu expression and with 38% of the tumors showing no change of
Her2/neu expression ( P = 0.037).
Conclusions: Using both IHC and FISH, advanced breast cancers show statistical evidence of decreasing incidence of Her2/neu expression
after antiaromatase neoadjuvant treatment. Our data also suggest that Her2/neu expression and its change during the treatment
might be predictive markers for this kind of therapy.
Cullin 4 (
CUL
4) and small ring finger protein
ROC
1 assemble to form E3 ubiquitin ligase (
CRL
4) complexes.
CUL
4 interacts with
WD
‐40 proteins through the adaptor protein
DNA
damage‐binding ...protein 1 (
DDB
1) to target substrates for ubiquitylation. Very little is known on how the
CUL
4 and
DDB
1 interaction is regulated. Here, we show that
DDB
1 is acetylated and acetylation promotes
DDB
1 binding to
CUL
4. We also identify nucleolar sirtuin 7 (
SIRT
7) as a major deacetylase that negatively regulates
DDB
1–
CUL
4 interaction. Following inhibition of nucleolar function by actinomycin D or 5‐fluorouracil treatment or knocking down the gene for the
RNA
polymerase I component
UBF
,
SIRT
7 is mobilized from the nucleolus to the nucleoplasm and promotes
DDB
1 deacetylation, leading to decreased
DDB
1–
CUL
4 association and
CRL
4 activity. This results in the accumulation or activation of
CRL
4 substrates including
LATS
1 and p73, which contribute to cell apoptosis induced by actinomycin D and 5‐fluorouracil. Our study uncovers a novel regulation of
CRL
4 E3 ligase complexes.
Chromosomal rearrangements involving telomeric bands have been frequently detected in many malignancies and congenital diseases. To develop a useful tool to study chromosomal rearrangements within ...the telomeric band effectively and accurately, a whole set of telomeric band painting probes (TBP) has been generated by chromosome microdissection. The intensity and specificity of these TBPs have been tested by fluorescence in situ hybridization and all TBPs showed strong and specific signals to target regions. TBPs of 6q and 17p were successfully used to detect the loss of the terminal band of 6q in a hepatocellular carcinoma cell line and a complex translocation involving the 17p terminal band in a melanoma cell line. Meanwhile, the TBP of 21q was used to detect a de novo translocation, t(12;21), and the breakpoint at 21q was located at 21q22.2. Further application of these TBPs should greatly facilitate the cytogenetic analysis of complex chromosome rearrangements involving telomeric bands.
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