Epigenetic regulation of human DCLK-1 gene during colon-carcinogenesis: clinical and mechanistic implications

Singh, Pomila; O’Connell, Malaney R.; Sarkar, Shubhashish

doi:10.21037/sci.2016.09.07

Cited by 28 publications

(30 citation statements)

References 45 publications

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“…DCLK1 has been shown be expressed in two forms, a full-length large form that encodes two microtubule-associating DCX domains at the N-terminus and a calmodulin-like kinase domain at the C-terminus. In addition, a shorter variant of the protein is expressed that lacks the N-terminal DCX domain [32][33][34][35] . To determine whether DCLK1 is expressed in OS cells, we performed western blot analyses of extracts generated from cells grown in 2-dimensional (2D) monolayer and 3-dimensional (3D) spheroid cultures.…”

Section: Pimozide Inhibits Stemnessmentioning

confidence: 99%

Suppressing STAT5 signaling affects osteosarcoma growth and stemness

et al. 2020

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Osteosarcoma (OS) is the most common primary bone tumor that primarily affects children and adolescents. Studies suggested that dysregulation JAK/STAT signaling promotes the development of OS. Cells treated with pimozide, a STAT5 inhibitor suppressed proliferation and colony formation and induced sub G0/G1 cell cycle arrest and apoptosis. There was a reduction in cyclin D1 and CDK2 expression and Rb phosphorylation, and activation of Caspase-3 and PARP cleavage. In addition, pimozide suppressed the formation of 3-dimensional osteospheres and growth of the cells in the Tumor in a Dish lung organoid system. Furthermore, there was a reduction in expression of cancer stem cell marker proteins DCLK1, CD44, CD133, Oct-4, and ABCG2. More importantly, it was the short form of DCLK1 that was upregulated in osteospheres, which was suppressed in response to pimozide. We further confirmed by flow cytometry a reduction in DCLK1+ cells. Moreover, pimozide inhibits the phosphorylation of STAT5, STAT3, and ERK in OS cells. Molecular docking studies suggest that pimozide interacts with STAT5A and STAT5B with binding energies of −8.4 and −6.4 Kcal/mol, respectively. Binding was confirmed by cellular thermal shift assay. To further understand the role of STAT5, we knocked down the two isoforms using specific siRNAs. While knockdown of the proteins did not affect the cells, knockdown of STAT5B reduced pimozide-induced necrosis and further enhanced late apoptosis. To determine the effect of pimozide on tumor growth in vivo, we administered pimozide intraperitoneally at a dose of 10 mg/kg BW every day for 21 days in mice carrying KHOS/NP tumor xenografts. Pimozide treatment significantly suppressed xenograft growth. Western blot and immunohistochemistry analyses also demonstrated significant inhibition of stem cell marker proteins. Together, these data suggest that pimozide treatment suppresses OS growth by targeting both proliferating cells and stem cells at least in part by inhibiting the STAT5 signaling pathway.

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Section: Pimozide Inhibits Stemnessmentioning

confidence: 99%

Suppressing STAT5 signaling affects osteosarcoma growth and stemness

et al. 2020

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“…Using ×10 ocular lenses and a ×2 Plan Apo objective (FOV = 10 mm) showed that 12 of the 70 CoAs measured ≤10 mm (mean 8.4 mm, range 7-10 mm), and the remaining 58 CoAs measured >11 mm (mean 16.1 mm, range 12-23 mm). The number of NECS in the 12 small CoA was 184 (mean 19.3, range 6-30), and the number of NECS in 14 unselected large CoAs was 267 (mean 20.1, range [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. The differences in the frequency of NECS between small and large CoAs was nonsignificant (p = 0.12).…”

Section: Frequency Of Necs In Small and Large Coamentioning

confidence: 99%

Disparate cell proliferation and p53 overexpression in colonic crypts with normal epithelial lining found below the neoplastic canopy of conventional adenomas

Rubio

Schmidt

2019

The Journal of Pathology CR

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We previously found colonic crypts with normal epithelial lining but with corrupted shapes (NECS) beneath the adenomatous tissue of conventional adenomas (CoAs). Here we assessed the distribution of proliferating cells (PCs) and explored the possible occurrence of p53‐upregulated cells in the NECS in a cohort of CoAs. Sections from 70 CoAs and from 12 normal colon segments were immunostained with the proliferation marker Ki67. In 60 of the 70 CoAs, additional sections were immunostained for the tumor suppressor p53 protein. NECS with asymmetric, haphazardly distributed single PC or PC clusters were recorded in 80% of the CoAs, with a continuous PC domain in one or both slopes of the crypts in 17%, and with haphazardly distributed single PCs in the remaining 3% of the CoAs. In the 12 normal segments (controls), the colon crypts demonstrated normal shapes with symmetric PC domains limited to the lower third portion of the crypts. In 30% of the 60 CoAs immunostained with p53 the NECS revealed haphazardly distributed p53‐upregulated cells, singly or in clusters. In sum, the apparently normal epithelium of the NECS beneath the adenomatous tissue of CoAs revealed an unprecedented relocation of the normal PC domains. This unexpected event and the occurrence of p53‐upregulated cells strongly suggest that the crypts beneath the neoplastic tissue of CoAs harbor somatic mutations. The accretion of putative mutated NECS beneath the neoplastic canopy of CoA emerges as a previously unaddressed major event, an event that might play an important role in the histogenesis of CoA in the human colon.

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“…3,4 Approximately 75% CC arises from long-term accumulation of epigenetic alterations. 5 In the past decades, a large number of epigenetic aberrations have been identified in CC patients, such as DNA methylation, histone modification, and expressions of microRNAs and long non-coding RNAs (lncRNAs). 6 Global DNA hypomethylation has been considered as a common epigenetic feature in CC patients.…”

Section: Introductionmentioning

confidence: 99%