https://openworks.mdanderson.org/sumexp21/1230/thumbnail.jp

Barkoudeh, Laila

Dhebat, Sarah

Ghosh, Bidyut

Maitra, Anirban

Shaheen, Menna

English

OpenWorks @ MD Anderson

BackgroundPancreatic cancer is the third leading cause of cancer death in both men and woman inthe USA and its incidence is progressively rising. Understanding the molecularunderpinnings of this lethal disease is crucial for developing potent therapies andeffective biomarkers. Studies have shown that pancreatic cancer is initiated as non-invasive ductal lesions that then progress to infiltrating pancreatic adenocarcinomathrough the accumulation of genetic alterations. Figure 1 shows the multistepprogression of pancreatic cancer from normal duct to infiltrating cancer. Thisprogression occurs through a series of histologically defined precursors calledPancreatic Intraepithelial Neoplasia (PanIN). They are precursors to pancreatic cancer,which is also known as pancreatic ductal adenocarcinoma (PDAC). In addition togenetic alterations, recent studies have shown how epigenetic modifications play animportant role in cancer progression as well. Unlike genetic modifications,epigenetics do not involve changes in DNA sequence, but rather affects geneexpression through reversible DNA or chromatin modifications, such as DNAmethylation and histone acetylation. Epigenetic regulation takes place through avariety of different enzymes also known as the writers, readers, and erasers (Figure 2).The writers add a variety of chemical groups onto DNA or histone amino acids, forexample histone lysine methylases can add up to three methyl groups onto one lysine.One of the well characterized lysine modifications is H3K9me3, a mark for closedchromatin (heterochromatin), which means it is inaccessible for gene expression i.e.gene expression is suppressed. The modifications that writers lay are not permanent,and can be reversed by another group of epigenetic modifiers known as erasers. Lysinedemethylases like KDM4C remove methyl groups from methylated lysines, resultingin open chromatin formation (euchromatin) and allowing gene expression to occur. Anexample is the histone demethylase KDM4C, an eraser in the Jumonji (JMJ) family.The Structure of KDM4C is shown in Figure 3, and it consists of two JMJ familydomains, one of which is responsible for the structural integrity and the second, whichis the catalytic domain. Recent studies suggest that the altered regulation of theseepigenetic tools plays a key role in tumorigenesis. Since these modifications arereversible and induced by deregulated enzymes, therapeutic approaches targeting thesealterations are areas of great interest in preclinical therapeutics. KDM4Coverexpression has been reported in lung, prostate, breast and brain cancers. KDM4Chas not been previously studied in pancreatic cancer. Therefore, we interrogated theexpression of KDM4C in pancreatic cancer and its effect on tumor growth.MethodsIn our laboratory we use orthogonal in-vitro and in-vivo model systems to study KDM4Cin PDAC. The in-vitro models are (i) human and mouse tissues for assessment of KDM4Cexpression, and (ii) human pancreatic cancer cell lines with CRISPR/Cas9 inducedknockout of endogenous KDM4C. For in-vivo analyses, we have athymic mice that areinjected with PDAC cells bearing knockout of KDM4C versus control lines, as well asnovel genetically engineered mouse models of PDAC with deregulated Kdm4c expression.Only the in vitro studies are described here, which were conducted by L.B. We firstsectioned human and mouse tissues and used multiplex IF technology to stain for KDM4Cexpression, including the epithelial marker EpCAM and neoplastic marker CK19 in themultiplex panel. Confocal microscopy was used to evaluate expression andphotomicrographs obtained. We then used CRISPR/Cas9 and guide RNAs against KDM4Cto knockout the endogenous protein in AsPC1 pancreatic cancer cell line. We conductedreal time PCR and western blot analysis for quantitative assessment of KDM4Cexpression. Finally colony formation assays were conducted to evaluate the impact ofKDM4C loss on AsPC1 anchorage independent growth.ConclusionsKDM4C is overexpressed in human and mouse pancreatic cancer tissues relative to normal pancreatic tissue. The downregulation of KDM4C expression significantly impedes anchorage independent growth of AsPC1 cells. These foundational data form the basis for future studies that will examine the downstream effector pathways of aberrant KDM4C expression in pancreatic cancer, including transcripts whose expression is altered as a result of changes in the H3K9me levels in the corresponding regulatory regions. We anticipate these future studies will also identify potential actionable pathways that might form the basis for reversing the oncogenic effects of KDM4C in pancreatic cancer. Referenceshttps://clincancerres.aacrjournals.org/content/6/8/2969.full-text.pdfhttps://pubmed.ncbi.nlm.nih.gov/31304427/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/Figure 4. Multiplex immunofluorescence of normal pancreas (A-D) and pancreaticductal adenocarcinoma (I-L) showing overexpression of Kdm4C protein (red) in aKras;p53 mouse model of pancreatic cancer. EpCam (green), an epithelial markerand CK19 (magenta), a marker of PDAC are also overexpressed. E-H, showselevated expression of Kdm4C in regions of acinar ductal metaplasia (ADM)compared to histologically normal pancreas.KDM4C is an Oncogenic Histone Demethylase in Pancreatic Cancer Laila Barkoudeh, Mennatallah Shaheen, M.S., Sarah Dhebat, M.S., Anirban Maitra, M.B.B.S., and Bidyut Ghosh, Ph.D. University of Texas MD Anderson Cancer Center, Houston, TexasFigure 1. Multistep progression model of pancreatic cancerFigure 2. Epigenetic modifiers - writers, readers and erasersFigure 3. KDM4C structure and domains Figure 5.  Western Blot showing low tp absent Kdm4c protein levels in single cell clones of AsPC1 cell line with CRISPR-induced KDM4C knockout, versus parental AsPC1 line. Actin is used as a loading control. Figure 5. Immunofluorescence of Kdm4C (red), EpCam (green) and Ck19(magenta) expression in resected normal (A-D) and cancer-bearing regions (E-L)of a representative patient-derived pancreatic cancer sample. Note the elevatedexpression of Kdm4C 9 in the PDAC region compared to the normal pancreas ofthe same patient. Localization of Kdm4c protein within the neoplastic epitheliumis confirmed by co-localization of EpCAM and CK19 expression.Figure 8. Colony Formation Assay.  Well A is AsPC1 control cell line. Wells BB,C, and D are the CRISPR knockouts KO1, KO2, and KO3 respectively.KDM4C is Overexpressed in Pancreatic Cancer Tissues Compared to Non-neoplastic Mouse and Human PancreasKDM4C Protein Levels Are Depleted After CRISPR KnockoutsKDM4C Relative Gene Expression in Control Cell Line vs KnockoutsFigure 6.  qRT-PCR shows depleted levels of Kdm4c transcripts in the three  AsPC1 knockouts vs control cell line.KO1KO2KO3Loss of KDM4C Reduces Anchorage Independent Growth ResultsPartnership for Careers in Cancer Science and Medicine

KDM4C is an Oncogenic Histone Demethylase in Pancreatic Cancer

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1230&context=sumexp21

KDM4C is an Oncogenic Histone Demethylase in Pancreatic Cancer

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