Rita Humeniuk

Carcinoma Associated Fibroblast Like Differentiation of Human Mesenchymal Stem Cells

Carcinoma-associated fibroblasts (CAF) have recently been implicated in important aspects of epithelial solid tumor biology, such as neoplastic progression, tumor growth, angiogenesis, and metastasis. However, neither the source of CAFs nor the differences between CAFs and fibroblasts from nonneoplastic tissue have been well defined. In this study, we show that human bone marrow-derived mesenchymal stem cells (hMSCs) exposed to tumor-conditioned medium (TCM) over a prolonged period of time assume a CAF-like myofibroblastic phenotype. More importantly, these cells exhibit functional properties of CAFs, including sustained expression of stromal-derived factor-1 (SDF-1) and the ability to promote tumor cell growth both in vitro and in an in vivo coimplantation model, and expression of myofibroblast markers, including alpha-smooth muscle actin and fibroblast surface protein. hMSCs induced to differentiate to a myofibroblast-like phenotype using 5-azacytidine do not promote tumor cell growth as efficiently as hMSCs cultured in TCM nor do they show increased SDF-1 expression. Furthermore, gene expression profiling revealed similarities between TCM-exposed hMSCs and CAFs. Taken together, these data suggest that hMSCs are a source of CAFs and can be used in the modeling of tumor-stroma interactions. To our knowledge, this is the first report showing that hMSCs become activated and resemble carcinoma-associated myofibroblasts on prolonged exposure to conditioned medium from MDAMB231 human breast cancer cells.

A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance.

MicroRNAs are predicted to regulate ≈30% of all human genes by targeting sequences in their 3′ UTR. Polymorphisms in 3′ UTR of several genes have been reported to affect gene expression, but the mechanism is not fully understood. Here, we demonstrate that 829C→T, a naturally occurring SNP, near the miR-24 binding site in the 3′ UTR of human dihydrofolate reductase (DHFR) affects DHFR expression by interfering with miR-24 function, resulting in DHFR overexpression and methotrexate resistance. miR-24 has a conserved binding site in DHFR 3′ UTR. DHFR with WT and 3′ UTR containing the 829C→T mutation were expressed in DG44 cells that lack DHFR. Overexpression of miR-24 in cells with WT DHFR resulted in down-regulation of DHFR protein, whereas no effect on DHFR protein expression was observed in the mutant 3′ UTR-expressing cells. Inhibition of endogenous miR-24 with a specific inhibitor led to up-regulation of DHFR in WT and not in mutant cells. Cells with the mutant 3′ UTR had a 2-fold increase in DHFR mRNA half-life, expressed higher DHFR mRNA and DHFR protein, and were 4-fold more resistant to methotrexate as compared with WT cells. SNP-829C→T, therefore, leads to a decrease in microRNA binding leading to overexpression of its target and results in resistance to methotrexate. We demonstrate that a naturally occurring miRSNP (a SNP located at or near a microRNA binding site in 3′ UTR of the target gene or in a microRNA) is associated with enzyme overproduction and drug resistance.

MiR-24 Tumor Suppressor Activity Is Regulated Independent of p53 and through a Target Site Polymorphism

MicroRNAs (miRNAs) are predicted to regulate approximately 30% of all human genes; however, only a few miRNAs have been assigned their targets and specific functions. Here we demonstrate that miR-24, a ubiquitously expressed miRNA, has an anti-proliferative effect independent of p53 function. Cell lines with differential p53 status were used as a model to study the effects of miR-24 on cell proliferation, cell cycle control, gene regulation and cellular transformation. Overexpression of miR-24 in six different cell lines, independent of p53 function, inhibited cell proliferation and resulted in G2/S cell cycle arrest. MiR-24 over expression in cells with wt-p53 upregulated TP53 and p21 protein; however, in p53-null cells miR-24 still induced cell cycle arrest without the involvement of p21. We show that miR-24 regulates p53-independent cellular proliferation by regulating an S-phase enzyme, dihydrofolate reductase (DHFR) a target of the chemotherapeutic drug methotrexate (MTX). Of interest, we found that a miR-24 target site polymorphism in DHFR 3′ UTR that results in loss of miR-24-function and high DHFR levels in the cell imparts a growth advantage to immortalized cells and induces neoplastic transformation. Of clinical significance, we found that miR-24 is deregulated in human colorectal cancer tumors and a subset of tumors has reduced levels of miR-24. A novel function for miR-24 as a p53-independent cell cycle inhibitory miRNA is proposed.

Decreased levels of UMP kinase as a mechanism of fluoropyrimidine resistance

5-Fluorouracil (5-FU) continues to be widely used for treatment of gastrointestinal cancers. Because many tumors show primary or acquired resistance, it is important to understand the molecular basis underlying the mechanism of resistance to 5-FU. In addition to its effect on thymidylate synthase inhibition and DNA synthesis, 5-FU may also influence RNA metabolism. Our previous studies revealed that colorectal cancer cells resistant to bolus 5-FU (HCT-8/4hFU) showed significantly decreased incorporation of the drug into RNA. Resistance to bolus 5-FU was associated with lower expression of UMP kinase (UMPK), an enzyme that plays an important role in the activation of 5-FU to 5-FUTP and its incorporation into RNA. Activities of other 5-FU–metabolizing enzymes (e.g., thymidine kinase, uridine phosphorylase, thymidine phosphorylase, and orotate phosphoribosyltransferase) remained unchanged between sensitive and resistant cell lines. Herein, we show that UMPK down-regulation in 5-FU–sensitive cells (HCT-8/P) induces resistance to bolus 5-FU treatment. Moreover, HCT-8/4hFU cells are even more cross-resistant to treatment with 5-fluorouridine, consistent with the current understanding of 5-fluorouridine as a RNA-directed drug. Importantly, colorectal cancer hepatic metastases isolated from patients clinically resistant to weekly bolus 5-FU/leucovorin treatment exhibited decreased mRNA expression of UMPK but not thymidylate synthase or dihydropyrimidine dehydrogenase compared with tumor samples of patients not previously exposed to 5-FU. Our findings provide new insights into the mechanisms of acquired resistance to 5-FU in colorectal cancer and implicate UMPK as an important mechanism of clinical resistance to pulse 5-FU treatment in some patients.[Mol Cancer Ther 2009;8(4):OF1–8]

Concise Review: Erythroid Versus Myeloid Lineage Commitment: Regulating the Master Regulators†‡§

Developmental processes, like blood formation, are orchestrated by transcriptional networks. Those transcriptional networks are highly responsive to various environmental stimuli and affect common precursors resulting in increased production of cells of the erythroid lineage or myeloid lineage (granulocytes, neutrophils, and macrophages). A significant body of knowledge has accumulated describing transcription factors that drive differentiation of these two major cellular pathways, in particular the antagonistic master regulators such as GATA‐1 and PU.1. However, little is known about factors that work upstream of master regulators to enhance differentiation toward one lineage. These functions become especially important under various stress conditions like sudden loss of red blood cells or pathogen infection. This review describes recent studies that begin to provide evidence for such factors. An increased understanding of factors regulating cellular commitment will advance our understanding of the etiology of diseases like anemia, cancer, and possibly other blood related disorders. STEM Cells2013;31:1237–1244

Epigenetic reversal of acquired resistance to 5-fluorouracil treatment

Acquired and intrinsic resistance still remains a limitation to the clinical use of 5-fluorouracil (5-FU). The contribution of epigenetic changes to the development of drug resistance remains to be elucidated. Several genes that are hypermethylated and silenced have been identified in colorectal cancer. Based on the findings described in the accompanying article, we hypothesized that acquired resistance to “pulse” 5-FU has an epigenetic origin and might be reversed. Here, we present a novel therapeutic approach to circumvent clinical resistance to bolus 5-FU, that is, treatment of bolus 5-FU-resistant colorectal cancer cells with low-dose 5-azadeoxycytidine (DAC), an inhibitor of DNA hypermethylation, restored sensitivity to 5-FU as well as 5-fluorouridine. Moreover, treatment of nude mice bearing a 5-FU-resistant tumor, characterized by decreased levels of UMP kinase (UMPK), with DAC overcame resistance to bolus 5-FU. DAC-mediated restoration of 5-FU sensitivity was associated with increases in UMPK levels. An increase in UMPK protein and mRNA levels following treatment with low-dose DAC was observed in cultured bolus 5-FU-resistant colorectal cancer cells (HCT-8) and in mice bearing these tumors. We conclude that DAC-mediated restoration of sensitivity to bolus 5-FU is mediated at least in part by increased UMPK levels and clinical resistance to 5-FU due to decreased UMPK in colorectal cancer may be overcome by including methylation inhibitors such as DAC. [Mol Cancer Ther 2009;8(5):1045–54]

Brief Report: Loss of p15Ink4b Accelerates Development of Myeloid Neoplasms in Nup98‐HoxD13 Transgenic Mice

Homeostasis of hematopoietic stem and progenitor cells is a tightly regulated process. The disturbance of the balance in the hematopoietic progenitor pool can result in favorable conditions for development of diseases such as myelodysplastic syndromes and leukemia. It has been shown recently that mice lacking p15Ink4b have skewed differentiation of common myeloid progenitors toward the myeloid lineage at the expense of erythroid progenitors. The lack of p15INK4B expression in human leukemic blasts has been linked to poor prognosis and increased risk of myelodysplastic syndromes transformation to acute myeloid leukemia. However, the role of p15Ink4b in disease development is just beginning to be elucidated. This study examines the collaboration of the loss of p15Ink4b with Nup98‐HoxD13 translocation in the development of hematological malignancies in a mouse model. Here, we report that loss of p15Ink4b collaborates with Nup98‐HoxD13 transgene in the development of predominantly myeloid neoplasms, namely acute myeloid leukemia, myeloproliferative disease, and myelodysplastic syndromes. This mouse model could be a very valuable tool for studying p15Ink4b function in tumorigenesis as well as preclinical drug testing. Stem Cells 2014;32:1361–1366

The tumor suppressor p15Ink4b regulates the differentiation and maturation of conventional dendritic cells.

The tumor suppressor p15Ink4b is frequently inactivated by methylation in acute myeloid leukemia and premalignant myeloid disorders. Dendritic cells (DCs) as potent APCs play critical regulatory roles in antileukemic immune responses. In the present study, we investigated whether p15Ink4b can function as modulator of DC development. The expression of p15Ink4b is induced strongly during differentiation and activation of DCs, and its loss resulted in significant quantitative and qualitative impairments of conventional DC (cDC) development. Accordingly, ex vivo-generated BM-derived DCs from p15Ink4b-knockout mice express significantly decreased levels of the antigen-presenting (MHC II) and costimulatory (CD80 and CD86) molecules and have impaired immunostimulatory functions, such as antigen uptake and T-cell stimulation. Reexpression of p15Ink4b in progenitors restored these defects, and confirmed a positive role for p15Ink4b during cDC differentiation and maturation. Furthermore, we have shown herein that p15Ink4b expression increases phosphorylation of Erk1/Erk2 kinases, which leads to an elevated activity of the PU.1 transcription factor. In conclusion, our results establish p15Ink4b as an important modulator of cDC development and implicate a novel function for this tumor suppressor in the regulation of adaptive immune responses.

Abstract 1973: Tumor suppressor p15Ink4b determines cell fate of hematopoietic progenitors: Implications for development of human blood disorders.

Red blood cells (RBCs) are a vital component of mammalian blood. Since they are short lived, they must be continuously replenished by erythropoiesis, a stepwise commitment of blood stem and progenitor cells to mature erythrocytes. The anemia due to the loss of RBC is a life threatening condition, often accompanying blood diseases such as leukemias and myelodysplastic syndromes (MDS). A striking 60-80% of these diseases have deleted or silenced expression of p15INK4B. An increased understanding of the factors that drive erythroid lineage commitment in progenitor cells is critical for developing new treatments for blood disorders. Previous examination of p15Ink4b knock-out mouse models, revealed skewing of hematopoietic progenitor differentiation towards myeloid lineage (granulocytes, macrophages). Here, we demonstrate a novel function for p15Ink4b in driving commitment to the erythroid lineage. Mice lacking p15Ink4b have lower numbers of primitive RBC progenitors and died shortly after induction of hemolytic anemia by phenylhydrazine injection. Expression of p15Ink4b in blood progenitors induced dynamic changes at the molecular level that rendered multi-lineage cells more permissive to erythroid commitment and less permissive to myeloid commitment. Noticeably, we found that p15Ink4b regulates a switch that controls the balance between myeloid and erythroid differentiation through activation of MEK/ERK signaling. In a time-coordinated manner expression of p15Ink4b induced rapid phosphorylation of MEK/ERK that led to rapid degradation of GATA-2 and activation of the GATA-1 transcription factors. Subsequently, the active GATA-1 executed lineage commitment through activation of the Erythropoietin receptor (EpoR), the “master regulator” of erythroid differentiation. The p15Ink4b mediated increase in GATA-1 expression, also resulted in decreased expression of the myeloid specific transcription factor PU.1, suppressing myeloid differentiation. In summary, we have defined a framework that determines how multipotent progenitors coordinate the balance between myeloid and erythroid differentiation. Central to this activity is p15Ink4b, which promotes erythroid fate while suppressing myeloid cell formation, a function that is particularly important in rapid RBC replenishment following stress. Our finding has implications not only for MDS and myeloid leukemia, where loss of tumor suppressor p15INK4B is a common event, but also for other forms of human refractory anemia. Citation Format: Rita Humeniuk, Michael Rosu-Myles, Linda Wolff. Tumor suppressor p15Ink4b determines cell fate of hematopoietic progenitors: Implications for development of human blood disorders. [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 1973. doi:10.1158/1538-7445.AM2013-1973