Cancer cells and stem cells share a number of biological characteristics including abundant amounts of decondensed chromatin. However, the molecular correlates and the factors involved in altering chromatin structure in cancer cells are not well known. Here, we report that less differentiated stem-like cells in the basal compartment of human and mouse prostate contain lower levels of the polycomb heterochromatin marker H3K27me3 than more differentiated luminal cells. This link to differentiated normal cells is also found in a number of other human and rodent tissues characterized by hierarchical differentiation. In addition to MYC's traditional role as a gene-specific transcription factor, recent studies indicate that MYC also affects global chromatin structure where it is required to maintain "open" or active chromatin. We now demonstrate that in both MYC-driven prostate cancers in mice and human prostate cancers, global levels of H3K27me3 are reduced in prostatic intraepithelial neoplasia and invasive adenocarcinoma lesions. Moreover, decreased levels of H3K27me3 correlate with increased markers of disease aggressiveness (eg, Gleason score and pathological stage). In vitro, experimentally forced reductions in MYC levels result in increased global levels of H3K27me3. These findings suggest that increased levels of decondensed chromatin in both normal progenitor cells and cancer cells are associated with global loss of H3K27me3, which is linked to MYC overexpression.
Abstract The polycomb gene, BMI1, has been reported to be critical in regulating self-renewal and proliferation of a number of tissue-specific normal as well as cancer stem cells, and it has been shown to be overexpressed in several tumor types. BMI1 has been reported to be highly expressed in the prostate basal cell compartment (Lukacs et al 2010, Cell Stem Cell 7: 682-693), which houses prostate stem cells (Robinson et al 1998, Prostate 37: 149-160), as well as being upregulated in prostate cancer (Lukacs et al 2010, van Leenders et al 2007, Eur Urol 52: 455-463). However, other studies have not found BMI1 levels to correlate with prostate cancer (Wolters et al 2010, BJU Int 106: 280-286). We used immunohistochemical staining as well as chromogenic in situ hybridization to characterize the pattern of BMI1 expression in normal prostate as well as primary and metastatic adenocarcinoma specimens using both standard glass slides and tissue microarrays. For our IHC staining, we used the antibody from Cell Signaling, and we validated that on human prostate tissue, a single band is obtained by western immunoblot. Using this antibody, we were unable to show any consistent difference in BMI1 protein between prostate basal and luminal cells, with generally abundant staining in both compartments. Similar findings were seen with in situ hybridization for BMI1 mRNA. We also found that BMI1 is not consistently upregulated in primary or metastatic human prostate cancers, albeit most tumor cells in most cases showed robust nuclear staining. The findings that BMI1 is not enriched in the stem cell compartment, does not label only a subset of prostate cancer cells, and is not generally upregulated in prostate cancer to any significant degree supports the concept that BMI1 would not be considered a marker of stem cells, cancer stem cells or a cancer biomarker in the prostate. Citation Format: Laxmi Gurunatham Pellakuru, Berrak Ocal-Gumuskaya, Qizhi Zheng, Jessica Hicks, Angelo De Marzo. BMI1 is not a marker of cancer or stem cells in the prostate. [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 4901. doi:10.1158/1538-7445.AM2013-4901
The ability of cells to respond and repair DNA damage is fundamental for the maintenance of genomic integrity. Ex vivo culturing of surgery-derived human tissues has provided a significant advancement to assess DNA damage response (DDR) in the context of normal cytoarchitecture in a non-proliferating tissue. Here, we assess the dependency of prostate epithelium DDR on ATM and DNA-PKcs, the major kinases responsible for damage detection and repair by nonhomologous end-joining (NHEJ), respectively. DNA damage was caused by ionizing radiation (IR) and cytotoxic drugs, cultured tissues were treated with ATM and DNA-PK inhibitors, and DDR was assessed by phosphorylation of ATM and its targets H2AX and KAP1, a heterochromatin binding protein. Phosphorylation of H2AX and KAP1 was fast, transient and fully dependent on ATM, but these responses were moderate in luminal cells. In contrast, DNA-PKcs was phosphorylated in both luminal and basal cells, suggesting that DNA-PK-dependent repair was also activated in the luminal cells despite the diminished H2AX and KAP1 responses. These results indicate that prostate epithelial cell types have constitutively dissimilar responses to DNA damage. We correlate the altered damage response to the differential chromatin state of the cells. These findings are relevant in understanding how the epithelium senses and responds to DNA damage.
Abstract Both embryonic stem cells and various cancer cell types contain abundant amounts of decondensed chromatin. However, the molecular mechanisms responsible for altering chromatin states in stem cells and cancer cells are not well understood. One factor may be the oncogenic transcription factor, MYC. MYC is overexpressed in many tumor types and affects a wide array of essential cellular processes including ribosome and mitochondrial biogenesis, metabolism, and cell cycle progression. MYC is also a key factor in reprogramming of somatic adult cells into induced pluripotent stem cells. Less well known is that MYC also affects global chromatin structure where it is required to maintain open or active chromatin. We hypothesize that in prostate cancer, MYC affects global chromatin structure keeping it in a more open conformation thus maintaining the cells in a less differentiated state resembling that of stem cells. We show by immunohistochemistry that compared to the more differentiated luminal cells, less differentiated stem-like cells in the basal compartment of the human and mouse prostate contain lower levels of the polycomb heterochromatin mark, H3K27me3. This link to the differentiated state of normal cells is also found in a number of other human and rodent tissues characterized by hierarchical differentiation and rapid turnover of stem cell compartments. Further, in MYC-driven prostate cancers in mice, global levels of H3K27me3 are reduced in both prostatic intraepithelial neoplasia (PIN) and invasive adenocarcinoma lesions. This pattern of global H3K27me3 reduction in PIN and invasive prostate cancers is also observed in humans, correlates with global chromatin decondensation, and correlates with increased markers of disease aggressiveness (e.g. Gleason score and pathological stage). In prostate and breast cancer cells, experimentally forced reductions in MYC levels result in increased global levels of H3K27me3. These findings suggest that increased levels of decondensed chromatin in normal stem/progenitor cells and in cancer cells are associated with global loss of the H3K27me3 polycomb mark, and furthermore, MYC overexpression could be one mechanism for this H3K27me3 deregulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2210. doi:1538-7445.AM2012-2210
Michael C. Haffner 1 , Alcides Chaux 2 , Alan K. Meeker 1,2,3 , David M. Esopi 1 , Jonathan Gerber 4 , Laxmi G. Pellakuru 2 , Antoun Toubaji 2 , Pedram Argani 1,2 , Christine Iacobuzio-Donahue 1,2 , William G. Nelson 1,2,3 , George J. Netto 1,2,3 , Angelo M. De Marzo 1,2,3 , Srinivasan Yegnasubramanian 1 1 Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland, USA 2 Department of Pathology, Johns Hopkins University, Baltimore, Maryland, USA 3 Brady Urological Institute, Johns Hopkins University, Baltimore, Maryland, USA 4 Department of Medicine, Division of Hematology, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA Received: September 1, 2011; Accepted: September 1, 2011; Published: September 2, 2011; Keywords: 5-hydroxymethylcytosine, 5hmC, DNA methylation, differentiation, cancer, tissue stem / progenitor cells Correspondence: Srinvasan Yegnasubramanian, email: // // Abstract DNA methylation at the 5-position of cytosines (5mC) represents an important epigenetic modification involved in tissue differentiation and is frequently altered in cancer. Recent evidence suggests that 5mC can be converted to 5-hydroxymethylcytosine (5hmC) in an enzymatic process involving members of the TET protein family. Such 5hmC modifications are known to be prevalent in DNA of embryonic stem cells and in the brain, but the distribution of 5hmC in the majority of embryonic and adult tissues has not been rigorously explored. Here, we describe an immunohistochemical detection method for 5hmC and the application of this technique to study the distribution of 5hmC in a large set of mouse and human tissues. We found that 5hmC was abundant in the majority of embryonic and adult tissues. Additionally, the level of 5hmC closely tracked with the differentiation state of cells in hierarchically organized tissues. The highest 5hmC levels were observed in terminally differentiated cells, while less differentiated tissue stem/progenitor cell compartments had very low 5hmC levels. Furthermore, 5hmC levels were profoundly reduced in carcinoma of the prostate, breast and colon compared to normal tissues. Our findings suggest a distinct role for 5hmC in tissue differentiation, and provide evidence for its large-scale loss in cancers.
On the Cover: In the pig model of cystic fibrosis (CF), mucous cell metaplasia and duct cell proliferation do not have a primary role in the pathogenesis of pancreatic disease but are a late response to fetal inflammation and tissue destruction.As shown in newborn pigs, obstructed ducts are often distended with mucus (magenta area), and the mucous cells are prominent in the epithelium.Cells were treated with PAS stain.(See page 504.
