Abstract The cytoplasmic enzyme cyclooxygenase (COX)-2 and its secondary lipid byproducts are critical determinants of cancer cell invasion and metastasis. Poorly differentiated MDA-MB-231 human breast cancer cells stably expressing COX-2 shRNA exhibit marked attenuation in their ability to secrete secondary lipid mediator products of the COX reaction, as well as invade and degrade reconstituted extracellular matrix in vitro. COX-2-silenced cells inoculated in vivo show significant reduction of tumor growth and extrapulmonary colonization than their parental counterpart. Based on compelling evidence for the positive correlation between cancer invasion and cell traction force, here we interrogated the mechanistic role for COX-2 in mediating this physical force and imaged the spatiotemporal distribution of contractile stress arising at the interface between each adherent cell and its substrate. Over a physiological range of substrate stiffness, COX-2-silenced MDA-MB-231 cells exhibited a smaller spreading area and a lower cell traction force than their highly metastatic, parental counterpart. Strikingly, unlike COX-2-silenced cells, COX-2-high parental MDA-MB-231 cells showed progressive increases in cell spreading area and traction force with increasing substrate rigidity. Cell spreading and traction force in COX-2-silenced cells were restored with an exogenous addition of COX-2-derived PGE2, however. COX-2-high MDA-MB-231 cells also showed increases in the expression of mechanosensitive integrin β1, and not β3, with increasing substrate rigidity. Consistent with these physical attributes, COX-2-silenced cells expressed a 4.6-fold reduction of the transcript coding for RhoJ, a member of the family of Rho GTPases, and displayed a slower remodeling dynamics of the cytoskeleton (CSK). To our knowledge this is the first report linking the expression of COX-2 with increased cell traction force and CSK remodeling in a highly metastatic human breast cancer. Citation Format: A Rum Yoon, Ioannis Stasinopoulos, Steven An, Zaver M. Bhujwalla. Mechano-molecular imaging of the role of COX-2 in cell traction force. [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 1098. doi:10.1158/1538-7445.AM2013-1098
Collaborator of ARF (CARF) was cloned as an ARF-interacting protein and shown to regulate the p53–p21WAF1–HDM2 pathway, which is central to tumor suppression via senescence and apoptosis. We had previously reported that CARF inhibition in cancer cells led to polyploidy and caspase-dependent apoptosis, however, the mechanisms governing this phenomenon remained unknown. Thus, we examined various cell death and survival pathways including the mitochondrial stress, ataxia telangiectasia mutated (ATM)–ATR, Ras–MAP kinase and retinoblastoma cascades. We found that CARF is a pleiotropic regulator with widespread effects; its suppression affected all investigated pathways. Most remarkably, it protected the cells against genotoxicity; CARF knockdown elicited DNA damage response as evidenced by increased levels of phosphorylated ATM and γH2AX, leading to induction of mitotic arrest and eventual apoptosis. We also show that the CARF-silencing-induced apoptosis in vitro translates to in vivo. In a human tumor xenograft mouse model, treatment of developing tumors with short hairpin RNA (shRNA) against CARF via an adenovirus carrier induced complete suppression of tumor growth, suggesting that CARF shRNA is a strong candidate for an anticancer reagent. We demonstrate that CARF has a vital role in genome preservation and tumor suppression and CARF siRNA is an effective novel cancer therapeutic agent.
MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21WAF1 (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3′UTR, and the Hu binding site of p21-3′UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21WAF1 pathway.
Abstract Background Emerging evidence supports that treatment of Crohn’s disease (CD) can alter the intestinal microbiota, which can play a significant role in the pathogenesis and therapeutic outcomes of CD. Ustekinumab (UST) has also been reported to modify the intestinal microbiota during a short-term treatment period. However, the long-term impact of UST treatment on the intestinal microbiota has rarely been studied. Methods We prospectively enrolled patients with CD who were planning to start UST treatment and collected fecal samples before (baseline) and after UST administration at week 20 and week 52. Fecal bacterial taxonomic diversity and composition were analyzed through 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing. The difference in the intestinal microbiota between clinical remitters and non-remitters defined by Crohn’s disease activity index (CDAI) was investigated, along with the longitudinal changes in the intestinal microbiota. Results A total of 124 fecal samples acquired from 43 patients were analyzed (median age 30 years, male 76.7%, median CDAI at baseline 183.2). Clinical remission was observed in 34.9% (n=15/43), 61.0% (n=25/41), and 60.0% (n=24/40) at baseline, week 20, and week 52, respectively. Shannon’s alpha diversity index increased significantly at week 20 and week 52 compared to baseline (both P<0.001). Compared to non-remitters, a significant increase of Shannon’s alpha diversity index was also observed among remitters (P=0.044) across all time points, while no significant difference was noted at each time point. The beta diversity analysis based on the Bray-Curtis distance revealed a significant difference among remitters at week 20 (P=0.027), however, there were no significant differences at other time points. Compared to the baseline, the abundance of the Firmicutes phylum was higher at week 20 (P=0.048) and 52 (P=0.019). The Lactobacillus genus (P=0.042), and Lactobacillus gasseri group (P=0.016) were significantly less abundant in remitters, while the Lachnospiraceae family (P=0.017) was more abundant in remitters. At week 20, Bifidobacterium genus (P=0.027) and Bifidobacterium pseudolongum group (P=0.040) were significantly more abundant in remitter while the Lactobacillus genus was less abundant (P=0.009) in remitter (Figure 1). Conclusion The alteration of fecal microbiota after UST treatment may have the potential to serve as a biomarker for predicting clinical remission. Financial Support This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1A2C2095096), the grant (2020IT0012) from the Asan Institute for Life Sciences, Seoul, Korea and the grant (CR 2017-4) from CELLTRION PHARM, Inc., Chungcheongbuk-do, Korea.
Studies on correlations between trough levels of adalimumab (TLAs) and levels of antibody to adalimumab (ATA levels) with combined mucosal and transmural healing as well as clinical remission in Crohn’s disease (CD) in non-Caucasians are still lacking. TLAs and ATA levels were measured using prospectively collected serum samples drawn from CD patients on adalimumab (ADL) maintenance therapy for more than 1 year at Asan Medical Center, South Korea, from August 2017 to July 2018. We analysed correlations between TLA/ATA levels and combined mucosal and transmural healing as well as clinical remission. TLAs/ATA levels according to concomitant immunomodulator use were also evaluated. This study included 189 serum samples drawn from 149 patients. Ninety-eight patients were males (65.8%). The median age at diagnosis of CD and at starting ADL was 21.0 years (interquartile range [IQR], 18.0–28.0) and 31.0 years (IQR, 23.0–37.5), respectively. Fifty patients (33.6%) have been previously exposed to infliximab. Clinical remission (Crohn’s disease activity index [CDAI] < 150) was observed in 77.8% (147/189 samples) and combined mucosal and transmural healing was observed in 16.2% (18/111 samples). TLAs differed significantly between two groups divided by a cut-off value of ATA as 4 µg/ml-eq (7.051 µg/ml [IQR 5.185–10.191] in ATI-negative samples [n = 182 {96.3%}] vs. 0.001 µg/ml [IQR 0.001–0.677] in ATI-positive samples [n = 39 {6.2%}], p < 0.001). TLAs showed significant differences between groups with or without combined mucosal and transmural healing (9.817 µg/ml [IQR 7.665–12.488] vs. 7.051 µg/ml [IQR 5.185–10.191], p = 0.010) but not between groups with or without clinical remission (7.891 µg/ml [IQR 5.477–10.835] vs. 6.786 µg/ml [IQR 4.080–11.031], p = 0.171). There was no difference in TLAs and ATA levels without/with concomitant immunomodulator use at the time of measuring TLAs/ATA levels, during induction period and continuously from induction period to the time of measuring TLAs/ATA levels (Table 1). Abstract P320 – P320 TLAs/ATA levels according to concomitant immunomodulator use. TLAs above 11.79, 12.00 and 14.76 µg/ml (area under the receiver-operating characteristic curve = 0.695) identified patients on deep healing with specificities of 85%, 90% and 95%, respectively. Abstract P320 – P320 TLAs/ATA levels according to concomitant immunomodulator use. TLAs above 11.79, 12.00 and 14.76 µg/ml (area under the receiver-operating characteristic curve = 0.695) identified patients on deep healing with specificities of 85%, 90% and 95%, respectively. TLAs better correlated with combined mucosal and transmural healing than clinical remission in Korean CD patients on ADL maintenance therapy. There was no difference in TLAs/ATA levels according to concomitant immunomodulator use.
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