William L. Berry

KDM4/JMJD2 Histone Demethylases: Epigenetic Regulators in Cancer Cells

Lysine methylation is one of the most prominent histone posttranslational modifications that regulate chromatin structure. Changes in histone lysine methylation status have been observed during cancer formation, which is thought to be a consequence of the dysregulation of histone lysine methyltransferases or the opposing demethylases. KDM4/JMJD2 proteins are demethylases that target histone H3 on lysines 9 and 36 and histone H1.4 on lysine 26. This protein family consists of three ~130-kDa proteins (KDM4A-C) and KDM4D/JMJD2D, which is half the size, lacks the double PHD and Tudor domains that are epigenome readers and present in the other KDM4 proteins, and has a different substrate specificity. Various studies have shown that KDM4A/JMJD2A, KDM4B/JMJD2B, and/or KDM4C/JMJD2C are overexpressed in breast, colorectal, lung, prostate, and other tumors and are required for efficient cancer cell growth. In part, this may be due to their ability to modulate transcription factors such as the androgen and estrogen receptor. Thus, KDM4 proteins present themselves as novel potential drug targets. Accordingly, multiple attempts are under way to develop KDM4 inhibitors, which could complement the existing arsenal of epigenetic drugs that are currently limited to DNA methyltransferases and histone deacetylases.

The JMJD2A demethylase regulates apoptosis and proliferation in colon cancer cells

JMJD2A is a transcriptional cofactor and enzyme that catalyzes demethylation of histone H3 lysines 9 and 36 and is overexpressed in human tumors, but its role in oncogenesis remains unclear. Here, we show that JMJD2A interacts with the tumor suppressor p53 both in vitro and in HCT116 colon cancer cells. Chromatin immunoprecipitation assays demonstrated that JMJD2A was recruited together with p53 to the promoter of the p21 cell cycle inhibitor upon stimulation with the DNA damaging agent, adriamycin. Downregulation of JMJD2A resulted in increased expression of p21 and of the pro‐apoptotic Puma protein, whereas levels of the anti‐apoptotic Bcl‐2 protein were decreased. Furthermore, JMJD2A knock‐down led to reduced HCT116, DLD‐1 and HT‐29 colon cancer cell proliferation, while overexpression of JMJD2A enhanced HCT116 proliferation in low serum media. Finally, JMJD2A depletion induced apoptosis in HCT116 cells and this effect was less pronounced in the absence of p53. Collectively, these data indicate that JMJD2A is a novel promoter of colon cancer cell proliferation and survival, which mediates its effects in p53‐dependent and ‐independent ways. JMJD2A may therefore be a valid target to sensitize tumor cells to chemotherapy‐induced cell death and growth suppression. J. Cell. Biochem. 113: 1368–1376, 2012.

Exosome-mediated microRNA signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (DHA)

BackgroundDocosahexaenoic acid (DHA) is a natural compound with anticancer and anti-angiogenesis activity that is currently under investigation as both a preventative agent and an adjuvant to breast cancer therapy. However, the precise mechanisms of DHA’s anticancer activities are unclear. It is understood that the intercommunication between cancer cells and their microenvironment is essential to tumor angiogenesis. Exosomes are extracellular vesicles that are important mediators of intercellular communication and play a role in promoting angiogenesis. However, very little is known about the contribution of breast cancer exosomes to tumor angiogenesis or whether exosomes can mediate DHA’s anticancer action.ResultsExosomes were collected from MCF7 and MDA-MB-231 breast cancer cells after treatment with DHA. We observed an increase in exosome secretion and exosome microRNA contents from the DHA-treated cells. The expression of 83 microRNAs in the MCF7 exosomes was altered by DHA (>2-fold). The most abundant exosome microRNAs (let-7a, miR-23b, miR-27a/b, miR-21, let-7, and miR-320b) are known to have anti-cancer and/or anti-angiogenic activity. These microRNAs were also increased by DHA treatment in the exosomes from other breast cancer lines (MDA-MB-231, ZR751 and BT20), but not in exosomes from normal breast cells (MCF10A). When DHA-treated MCF7 cells were co-cultured with or their exosomes were directly applied to endothelial cell cultures, we observed an increase in the expression of these microRNAs in the endothelial cells. Furthermore, overexpression of miR-23b and miR-320b in endothelial cells decreased the expression of their pro-angiogenic target genes (PLAU, AMOTL1, NRP1 and ETS2) and significantly inhibited tube formation by endothelial cells, suggesting that the microRNAs transferred by exosomes mediate DHA’s anti-angiogenic action. These effects could be reversed by knockdown of the Rab GTPase, Rab27A, which controls exosome release.ConclusionsWe conclude that DHA alters breast cancer exosome secretion and microRNA contents, which leads to the inhibition of angiogenesis. Our data demonstrate that breast cancer exosome signaling can be targeted to inhibit tumor angiogenesis and provide new insight into DHA’s anticancer action, further supporting its use in cancer therapy.

Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1

BackgroundDoublecortin-like kinase 1 (DCLK1) is emerging as a tumor specific stem cell marker in colorectal and pancreatic cancer. Previous in vitro and in vivo studies have demonstrated the therapeutic effects of inhibiting DCLK1 with small interfering RNA (siRNA) as well as genetically targeting the DCLK1+ cell for deletion. However, the effects of inhibiting DCLK1 kinase activity have not been studied directly. Therefore, we assessed the effects of inhibiting DCLK1 kinase activity using the novel small molecule kinase inhibitor, LRRK2-IN-1, which demonstrates significant affinity for DCLK1.ResultsHere we report that LRRK2-IN-1 demonstrates potent anti-cancer activity including inhibition of cancer cell proliferation, migration, and invasion as well as induction of apoptosis and cell cycle arrest. Additionally we found that it regulates stemness, epithelial-mesenchymal transition, and oncogenic targets on the molecular level. Moreover, we show that LRRK2-IN-1 suppresses DCLK1 kinase activity and downstream DCLK1 effector c-MYC, and demonstrate that DCLK1 kinase activity is a significant factor in resistance to LRRK2-IN-1.ConclusionsGiven DCLK1’s tumor stem cell marker status, a strong understanding of its biological role and interactions in gastrointestinal tumors may lead to discoveries that improve patient outcomes. The results of this study suggest that small molecule inhibitors of DCLK1 kinase should be further investigated as they may hold promise as anti-tumor stem cell drugs.

Elevated Circulation Levels of an Antiangiogenic SERPIN in Patients with Diabetic Microvascular Complications Impair Wound Healing through Suppression of Wnt Signaling

Wound healing, angiogenesis and hair follicle maintenance are often impaired in the skin of diabetic patients, but the pathogenesis has not been well understood. Here, we report that circulation levels of kallistatin, a member of the serine proteinase inhibitor (SERPIN) superfamily with anti-angiogenic activities, were elevated in Type 2 diabetic patients with diabetic vascular complications. To test the hypothesis that elevated kallistatin levels could contribute to a wound healing deficiency via inhibition of Wnt/β-catenin signaling, we generated kallistatin-transgenic (KS-TG) mice. KS-TG mice had reduced cutaneous hair follicle density, microvascular density, and panniculus adiposus layer thickness as well as altered skin microvascular hemodynamics and delayed cutaneous wound healing. Using Wnt reporter mice, our results showed that Wnt/β-catenin signaling is suppressed in dermal endothelium and hair follicles in KS-TG mice. Lithium, a known activator of β-catenin via inhibition of glycogen synthase kinase-3β, reversed the inhibition of Wnt/β-catenin signaling by kallistatin and rescued the wound healing deficiency in KS-TG mice. These observations suggest that elevated circulating anti-angiogenic serpins in diabetic patients may contribute to impaired wound healing through inhibition of Wnt/β-catenin signaling. Activation of Wnt/β-catenin signaling, at a level downstream of Wnt receptors, may ameliorate the wound healing deficiency in diabetic patients.

Development and characterization of a preclinical model of breast cancer lung micrometastatic to macrometastatic progression.

Most cancer patients die with metastatic disease, thus, good models that recapitulate the natural process of metastasis including a dormancy period with micrometastatic cells would be beneficial in developing treatment strategies. Herein we report a model of natural metastasis that balances time to complete experiments with a reasonable dormancy period, which can be used to better study metastatic progression. The basis for the model is a 4T1 triple negative syngeneic breast cancer model without resection of the primary tumor. A cell titration from 500 to 15,000 GFP tagged 4T1 cells implanted into fat pad number four of immune proficient eight week female BALB/cJ mice optimized speed of the model while possessing metastatic processes including dormancy and beginning of reactivation. The frequency of primary tumors was less than 50% in animals implanted with 500–1500 cells. Although implantation with over 10,000 cells resulted in 100% primary tumor development, the tumors and macrometastases formed were highly aggressive, lacked dormancy, and offered no opportunity for treatment. Implantation of 7,500 cells resulted in >90% tumor take by 10 days; in 30–60 micrometastases in the lung (with many animals also having 2–30 brain micrometastases) two weeks post-implantation, with the first small macrometastases present at five weeks; many animals displaying macrometastases at five weeks and animals becoming moribund by six weeks post-implantation. Using the optimum of 7,500 cells the efficacy of a chemotherapeutic agent for breast cancer, doxorubicin, given at its maximal tolerated dose (MTD; 1 mg/kg weekly) was tested for an effect on metastasis. Doxorubicin treatment significantly reduced primary tumor growth and lung micrometastases but the number of macrometastases at experiment end was not significantly affected. This model should prove useful for development of drugs to target metastasis and to study the biology of metastasis.

Stimulation of β-catenin and colon cancer cell growth by the KDM4B histone demethylase

The linchpin of colorectal cancer is the oncoprotein and transcriptional cofactor β-catenin, whose overexpression is causative for the neoplastic transformation of colon cells. However, the molecular details of β-catenin dependent gene transcription in cancer cells are still not comprehensively explored. Here, we show that the histone demethylase KDM4B was upregulated in colon and rectal adenocarcinomas and required for efficient growth and clonogenic activity of human HT-29 colon cancer cells. Moreover, KDM4B formed complexes with β-catenin in vitro and in vivo, which involved its central amino acids 353-740. In addition, KDM4B also interacted with the DNA-binding protein TCF4, which is the main factor recruiting β-catenin to chromatin in the intestine. KDM4B downregulation resulted in reduced expression of the β-catenin/TCF4 target genes JUN, MYC and Cyclin D1, all of which encode for oncoproteins. Collectively, our data indicate that KDM4B overexpression supports β-catenin mediated gene transcription and thereby contributes to the genesis of colorectal tumors. Accordingly, inhibition of the KDM4B histone demethylase may represent a novel avenue of fighting colorectal cancer, one of the major causes of cancer death throughout the world.

Gene expression analysis of biological systems driving an organotypic model of endometrial carcinogenesis and chemoprevention

An organotypic model of endometrial carcinogenesis and chemoprevention was developed in which normal endometrial organotypic cultures exposed to the carcinogen, DMBA (7,12-dimethylbenz[a]anthracene), developed a cancerous phenotype in the absence, but not presence of subsequent treatment with a flexible heteroarotinoid (Flex-Het), called SHetA2. A discriminant function based on karyometric features of cellular nuclei and an agar clonogenic assay confirmed these histologic changes. Interpretation of microarray data using an internal standard approach identified major pathways associated with carcinogenesis and chemoprevention governed by c-myc, p53, TNFα and Jun genes. Cluster analysis of functional associations of hypervariable genes demonstrated that carcinogenesis is accompanied by a stimulating association between a module of genes that includes tumor necrosis factor α (TNFα), c-myc, and epidermal growth factor-receptor (EGF-R) and a module that includes insulin-like growth factor I-receptor (IGF-IR), p53, and Jun genes. Two secreted proteins involved in these systems, tenascin C and inhibin A, were validated at the protein level. Tenascin C is an EGF-R ligand, and therefore may contribute to the increased EGF-R involvement in carcinogenesis. The known roles of the identified molecular systems in DMBA and endometrial carcinogenesis and chemoprevention supports the validity of this model and the potential clinical utility of SHetA2 in chemoprevention.

Fluvastatin Interferes with Hepatitis C Virus Replication via Microtubule Bundling and a Doublecortin-like Kinase-Mediated Mechanism

Hepatitis C virus (HCV)-induced alterations in lipid metabolism and cellular protein expression contribute to viral pathogenesis. The mechanism of pleiotropic actions of cholesterol-lowering drugs, statins, against HCV and multiple cancers are not well understood. We investigated effects of fluvastatin (FLV) on microtubule-associated and cancer stem cell marker (CSC), doublecortin-like kinase 1 (DCLK1) during HCV-induced hepatocarcinogenesis. HCV replication models, cancer cell lines and normal human hepatocytes were used to investigate the antiviral and antitumor effects of statins. FLV treatment resulted in induction of microtubule bundling, cell-cycle arrest and alterations in cellular DCLK1 distribution in HCV-expressing hepatoma cells. These events adversely affected the survival of liver-derived tumor cells without affecting normal human hepatocytes. FLV downregulated HCV replication in cell culture where the ATP pool and cell viability were not compromised. Pravastatin did not exhibit these effects on HCV replication, microtubules and cancer cells. The levels of miR-122 that regulates liver homeostasis and provides HCV genomic stability remained at steady state whereas DCLK1 mRNA levels were considerably reduced during FLV treatment. We further demonstrated that HCV replication was increased with DCLK1 overexpression. In conclusion, unique effects of FLV on microtubules and their binding partner DCLK1 are likely to contribute to its anti-HCV and antitumor activities in addition to its known inhibitory effects on 3-hydroxy-3-methylglutary-CoA reductase (HMGCR).

PDGFRα controls the balance of stromal and adipogenic cells during adipose tissue organogenesis.

Adipose tissue is distributed in depots throughout the body with specialized roles in energy storage and thermogenesis. PDGFRα is a marker of adipocyte precursors, and increased PDGFRα activity causes adipose tissue fibrosis in adult mice. However, the function of PDGFRα during adipose tissue organogenesis is unknown. Here, by analyzing mice with juxtamembrane or kinase domain point mutations that increase PDGFRα activity (V561D or D842V), we found that PDGFRα activation inhibits embryonic white adipose tissue organogenesis in a tissue-autonomous manner. By lineage tracing analysis, we also found that collagen-expressing precursor fibroblasts differentiate into white adipocytes in the embryo. PDGFRα inhibited the formation of adipocytes from these precursors while favoring the formation of stromal fibroblasts. This imbalance between adipocytes and stromal cells was accompanied by overexpression of the cell fate regulator Zfp521. PDGFRα activation also inhibited the formation of juvenile beige adipocytes in the inguinal fat pad. Our data highlight the importance of balancing stromal versus adipogenic cell expansion during white adipose tissue development, with PDGFRα activity coordinating this crucial process in the embryo. Summary: Analysis of PDGFRα gain-of-function mutant mice demonstrates a role in balancing the expansion of fibroblast and adipocyte lineages before birth.