cyclin-d1 and Gastrointestinal-Stromal-Tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. The angiotensin II type 1 receptor blockers, telmisartan and candesartan, are widely used antihypertensive drugs that inhibits cancer cell proliferation; however, its underlying mechanisms in mesenchymal tumors, including GIST, remains unknown. The present study aimed to investigate the effect of telmisartan on GIST‑T1 cells and its underlying mechanism. Telmisartan and candesartan inhibited the proliferation of these cells by blocking the G0 to G1 cell cycle transition, which was accompanied by a decrease in cell cycle‑related proteins such as cyclin D1. Furthermore, telmisartan exposure significantly altered microRNA expression in vitro. In conclusion, telmisartan suppressed human GIST cell proliferation by inducing cell cycle arrest in vitro.

Topics: AMP-Activated Protein Kinases; Angiogenesis Inducing Agents; Angiotensin II Type 1 Receptor Blockers; Antihypertensive Agents; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gastrointestinal Neoplasms; Gastrointestinal Stromal Tumors; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Telmisartan; Tissue Array Analysis

Oncogenic KIT or PDGFRA tyrosine kinase mutations are compelling therapeutic targets in most gastrointestinal stromal tumors (GISTs), and the KIT inhibitor, imatinib, is therefore standard of care for patients with metastatic GIST. However, some GISTs lose expression of KIT oncoproteins, and therefore become KIT-independent and are consequently resistant to KIT-inhibitor drugs. We identified distinctive biologic features in KIT-independent, imatinib-resistant GISTs as a step towards identifying drug targets in these poorly understood tumors. We developed isogenic GIST lines in which the parental forms were KIT oncoprotein-dependent, whereas sublines had loss of KIT oncoprotein expression, accompanied by markedly downregulated expression of the GIST biomarker, protein kinase C-theta (PRKCQ). Biologic mechanisms unique to KIT-independent GISTs were identified by transcriptome sequencing, qRT-PCR, immunoblotting, protein interaction studies, knockdown and expression assays, and dual-luciferase assays. Transcriptome sequencing showed that cyclin D1 expression was extremely low in two of three parental KIT-dependent GIST lines, whereas cyclin D1 expression was high in each of the KIT-independent GIST sublines. Cyclin D1 inhibition in KIT-independent GISTs had anti-proliferative and pro-apoptotic effects, associated with Rb activation and p27 upregulation. PRKCQ, but not KIT, was a negative regulator of cyclin D1 expression, whereas JUN and Hippo pathway effectors YAP and TAZ were positive regulators of cyclin D1 expression. PRKCQ, JUN, and the Hippo pathway coordinately regulate GIST cyclin D1 expression. These findings highlight the roles of PRKCQ, JUN, Hippo, and cyclin D1 as oncogenic mediators in GISTs that have converted, during TKI-therapy, to a KIT-independent state. Inhibitors of these pathways could be effective therapeutically for these now untreatable tumors.

Topics: Antineoplastic Agents; Cell Proliferation; Cyclin D1; Gastrointestinal Stromal Tumors; Humans; Protein Kinase C-theta; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-kit

Non-random gains of chromosome 5p have been observed in clinically aggressive gastrointestinal stromal tumors, whereas the driving oncogenes on 5p remain to be characterized. We used an integrative genomic and functional approach to identify amplified oncogenes on 5p and to evaluate the relevance of AMACR amplification at 5p13.3 and its overexpression in gastrointestinal stromal tumors. Thirty-seven tumor samples, imatinib-sensitive GIST882 cell line, and imatinib-resistant GIST48 cell line were analyzed for DNA imbalances using array-based genomic profiling. Forty-one fresh tumor samples of various risk categories were enriched for pure tumor cells by laser capture microdissection and quantified for AMACR mRNA expression. AMACR-specific fluorescence in situ hybridization and immunohistochemistry were both informative in tissue microarray sections of 350 independent primary gastrointestinal stromal tumors, including 213 cases with confirmed KIT /PDGFRA genotypes. To assess the oncogenic functions of AMACR, GIST882 and GIST48 cell lines were stably silenced against their endogenous AMACR expression. In 59% of cases featuring 5p gains, two major amplicons encompassed discontinuous chromosomal regions that were differentially overrepresented in high-risk cases, including the one harboring the mRNA-upregulated AMACR gene. Gene amplification was detected in 19.7% of cases (69/350) and strongly related to protein overexpression (p<0.001), although 52% of AMACR-overexpressing cases exhibited no amplification. Both gene amplification and protein overexpression were significantly associated with epithelioid histology, larger size, increased mitoses, higher risk levels, and unfavorable genotypes (all p≦0.03). They were also independently predictive of decreased disease-free survival (overexpression, p<0.001; amplification, p=0.020) in the multivariate analysis. Concomitant with downregulated cyclin D1, cyclin E, and CDK4, AMACR knockdown suppressed cell proliferation and induced G1-phase arrest, but did not affect apoptosis in both GIST882 and GIST48 cells. In conclusion, AMACR amplification is a mechanism driving increased mRNA and protein expression and conferring aggressiveness through heightened cell proliferation in gastrointestinal stromal tumors.

Topics: Azoles; Benzamides; Cell Line, Tumor; Cell Proliferation; Comparative Genomic Hybridization; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 4; Disease-Free Survival; DNA Mutational Analysis; Flow Cytometry; G1 Phase; Gastrointestinal Stromal Tumors; Gene Dosage; Genotype; Humans; Imatinib Mesylate; Immunohistochemistry; In Situ Hybridization, Fluorescence; Isoindoles; Multivariate Analysis; Oncogene Proteins; Organoselenium Compounds; Piperazines; Predictive Value of Tests; Prognosis; Pyrimidines; Racemases and Epimerases; RNA Interference; Treatment Outcome; Up-Regulation

Gastrointestinal stromal tumor (GIST) is characterized by KIT or PDGFRA gene mutation. Although chromosomal losses of 22q are frequent in GIST, it is unclear which tumor suppressor genes might be inactivated in association with such losses. The INI1 gene, located at 22q11.23, is a tumor suppressor gene that is frequently altered in malignant rhabdoid tumor.. To elucidate the hypothesis that the INI1 gene might be altered along with 22q loss in GIST, we examined the loss of heterozygosity (LOH) at 22q11.23, homozygous deletion and mutation of the INI1 gene, and its gene product expression as well as mutations of KIT and PDGFRA in 27 cases of GIST.. Among the 27 informative cases, 19 (70.4%) showed LOH of at least one of the microsatellite markers on 22q11.23. None of the cases (0%) showed homozygous deletion or mutation of the INI1 gene. Immunohistochemically, the INI1 expression was focally reduced in 17/27 (63%) cases, and the INI1 protein level and INI1 mRNA level were each correlated with the presence of 22q11.23 LOH. Although the 22q11.23 LOH was more frequently present in high- than in low-grade tumors, INI1 expression level was not correlated with tumor grade, tumor size, proliferative activity and the expression levels of cyclin D1 and p16INK4a. KIT mutations were found in 18/27 (66.7%) GISTs; however, the KIT genotype was not correlated with the status of LOH at 22q11.23.. The results suggest that 22q11.23 LOH is frequently present in GIST irrespective of KIT genotype and it might play a role in part of the development of GIST. However, the hemiallelic loss of INI1 gene causing reduced expression of INI1 protein probably does not have a major impact in the progression of GIST.

Topics: Adult; Aged; Aged, 80 and over; Chromosomal Proteins, Non-Histone; Chromosomes, Human, Pair 22; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Disease Progression; DNA Mutational Analysis; DNA-Binding Proteins; Female; Gastrointestinal Stromal Tumors; Genotype; Humans; Loss of Heterozygosity; Male; Microsatellite Repeats; Middle Aged; Mutation; Proto-Oncogene Proteins c-kit; Receptor, Platelet-Derived Growth Factor alpha; RNA, Messenger; SMARCB1 Protein; Transcription Factors

CD44 and osteopontin (OPN) are functionally related molecules that, alone or in combination, play miscellaneous biological and pathophysiologic roles. CD44 cleavage, one unique feature of CD44, occurs in human cancers, but its function remains unclear. This study aimed to assess the clinicopathologic significance and mechanism of CD44 cleavage in gastrointestinal stromal tumor (GIST) with respect to OPN and OPN/CD44 interaction.. CD44 cleavage was evaluated by immunoblotting in 31 primary GIST tumor specimens with paired normal tissues. OPN/CD44 interaction was examined by in situ proximity ligation assay. The associations of CD44 cleavage activity with clinicopathologic parameters, cyclin D1 expression, beta-catenin expression, OPN expression, and OPN/CD44 interaction were analyzed.. CD44 cleavage activity was demonstrated in 87.1% of GIST, in contrast to its absence in normal tissues. Increased CD44 cleavage activity was significantly associated with enhanced mitosis by multivariate analysis, in addition to being related to tumor size, recurrence, high-risk status, and poor survival by univariate analysis. Mitosis was significantly higher in GIST with increased CD44 cleavage activity, which also positively correlated with tumor-specific beta-catenin and cyclin D1 overexpression, indicating a mitotic effect through aberrant cell cycle. Both OPN and OPN/CD44 interactions were significantly associated with CD44 cleavage.. Our study demonstrates the clinicopathological significance of CD44 cleavage in GIST. There is a significantly increased mitosis associated with CD44 cleavage in relation to OPN/CD44 interaction and dysregulated cell cycle in GIST.

Topics: Aged; beta Catenin; Blotting, Western; Cyclin D1; Female; Follow-Up Studies; Gastrointestinal Stromal Tumors; Humans; Hyaluronan Receptors; Male; Middle Aged; Mitosis; Osteopontin; Prognosis

To investigate the expression of p27 and cyclin D1 protein in gastrointestinal stromal tumors (GIST), and discuss the relationship between their expression and clincopathological characteristics.. Immunohistochemical staining was used to detect the expression of p27 and cyclin D1 in 48 cases of GIST and corresponding normal tissues.. In 48 cases GIST, the expression of p27 and cyclin D1 protein were 54.16% and 58.33% respectively. The low level expression of p27 protein was inversely correlated with the degree of differentiation and tumor recurrence of GIST (both P < 0.01). The expression of p27 protein was positively relatied to that of cyclin D1 (r = 0.573, P < 0.01).. These data shows the expression of p27 and cyclin D1 expression are related to the ontogenesis and progression of GIST. p27 and cyclin D1 may be prognostic indicators in GIST.

Topics: Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Gastrointestinal Stromal Tumors; Humans; Immunohistochemistry; Male; Middle Aged; Prognosis; Protein Kinase Inhibitors

To investigate the clinicopathologic features of gastrointestinal stromal tumors (GISTs) and to identify reliable prognostic parameters.. Fifty-nine GISTs were studied by immunostaining of CD117, CD34, SMA, desmin, S-100, bcl-2, and Ki-67. Histopathologic evaluations included tumor size, necrosis, histological growth patterns, mitotic activities and tumor lymphocytic infiltrate. The patients were clinically followed for 2 to 9 years. Univariate, multivariate and correlative statistical evaluations were used to analyze the data.. Among the 59 patients, 40 were alive and 15 died of their tumors at follow-up, the remaining 4 patients died of other causes. Pathological parameters that correlated with prognosis included tumor sizes of more than 5 cm, tumor tissue necrosis, mitotic cell count equal or higher than 5 per 50 high power field, Ki-67 labeling index (LI) equal or higher than 5% and intense bcl-2 immunostaining. Multivariate analysis showed that the mitotic count and Ki-67 LI were independent prognostic indicators. There was a correlation between mitotic count and Ki-67 LI.. Mitotic count and Ki-67 LI are the best predictors for a poor outcome of GIST after surgical treatment. Ki-67 immunostaining may substitute mitotic count as a useful prognostic parameter.

Topics: Adolescent; Adult; Aged; Child; Cyclin D1; Female; Follow-Up Studies; Gastrointestinal Stromal Tumors; Humans; Ki-67 Antigen; Male; Middle Aged; Prognosis; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-kit