Cheryl M. Koh

NOTCH pathway blockade depletes CD133-positive glioblastoma cells and inhibits growth of tumor neurospheres and xenografts

Cancer stem cells (CSCs) are thought to be critical for the engraftment and long‐term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem‐like cells with higher NOTCH activity. We therefore used low‐passage and established GBM‐derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by γ‐secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug‐impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem‐like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem‐like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas. STEM CELLS 2010;28:5–16

Nuclear MYC Protein Overexpression is an Early Alteration in Human Prostate Carcinogenesis

The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, protein synthesis, mitochondrial function and stem cell renewal. A region on chromosome 8q24 encompassing the MYC locus is amplified in prostate cancer, but this occurs mostly in advanced disease suggesting that MYC alterations occur late in prostate cancer. In contrast, MYC mRNA is elevated in most prostate cancers, even those of relatively low stage and grade (eg Gleason score 6) suggesting that MYC plays a role in initiation. However, since MYC protein levels are tightly regulated, elevated MYC mRNA does not necessarily imply elevated MYC protein. Thus, it is critical to determine whether MYC protein is elevated in human prostate cancer, and if so, at what stage of the disease this elevation occurs. Prior studies of MYC protein localization have been hampered by lack of suitable antibodies and controls. We utilized a new anti-MYC antibody coupled with genetically defined control experiments to localize MYC protein within human tissue microarrays consisting of normal, atrophy, PIN, primary adenocarcinoma, and metastatic adenocarcinoma. Nuclear overexpression of MYC protein occurred frequently in luminal cells of PIN, as well as in most primary carcinomas and metastatic disease. MYC protein did not correlate with gain of 8q24, suggesting alternative mechanisms for MYC overexpression. These results provide evidence that upregulation of nuclear MYC protein expression is a highly prevalent and early change in prostate cancer and suggest that increased nuclear MYC may be a critical oncogenic event driving human prostate cancer initiation and progression.

MYC and Prostate Cancer

Prostate cancer, the majority of which is adenocarcinoma, is the most common epithelial cancer affecting a majority of elderly men in Western nations. Its manifestation, however, varies from clinically asymptomatic insidious neoplasms that progress slowly and do not threaten life to one that is highly aggressive with a propensity for metastatic spread and lethality if not treated in time. A number of somatic genetic and epigenetic alterations occur in prostate cancer cells. Some of these changes, such as loss of the tumor suppressors PTEN and p53, are linked to disease progression. Others, such as ETS gene fusions, appear to be linked more with early phases of the disease, such as invasion. Alterations in chromosome 8q24 in the region of MYC have also been linked to disease aggressiveness for many years. However, a number of recent studies in human tissues have indicated that MYC appears to be activated at the earliest phases of prostate cancer (e.g., in tumor-initiating cells) in prostatic intraepithelial neoplasia, a key precursor lesion to invasive prostatic adenocarcinoma. The initiation and early progression of prostate cancer can be recapitulated in genetically engineered mouse models, permitting a richer understanding of the cause and effects of loss of tumor suppressors and activation of MYC. The combination of studies using human tissues and mouse models paints an emerging molecular picture of prostate cancer development and early progression. This picture reveals that MYC contributes to disease initiation and progression by stimulating an embryonic stem cell-like signature characterized by an enrichment of genes involved in ribosome biogenesis and by repressing differentiation. These insights pave the way to potential novel therapeutic concepts based on MYC biology.

Cell-Type Independent MYC Target Genes Reveal a Primordial Signature Involved in Biomass Accumulation

The functions of key oncogenic transcription factors independent of context have not been fully delineated despite our richer understanding of the genetic alterations in human cancers. The MYC oncogene, which produces the Myc transcription factor, is frequently altered in human cancer and is a major regulatory hub for many cancers. In this regard, we sought to unravel the primordial signature of Myc function by using high-throughput genomic approaches to identify the cell-type independent core Myc target gene signature. Using a model of human B lymphoma cells bearing inducible MYC, we identified a stringent set of direct Myc target genes via chromatin immunoprecipitation (ChIP), global nuclear run-on assay, and changes in mRNA levels. We also identified direct Myc targets in human embryonic stem cells (ESCs). We further document that a Myc core signature (MCS) set of target genes is shared in mouse and human ESCs as well as in four other human cancer cell types. Remarkably, the expression of the MCS correlates with MYC expression in a cell-type independent manner across 8,129 microarray samples, which include 312 cell and tissue types. Furthermore, the expression of the MCS is elevated in vivo in Eμ-Myc transgenic murine lymphoma cells as compared with premalignant or normal B lymphocytes. Expression of the MCS in human B cell lymphomas, acute leukemia, lung cancers or Ewing sarcomas has the highest correlation with MYC expression. Annotation of this gene signature reveals Mycs primordial function in RNA processing, ribosome biogenesis and biomass accumulation as its key roles in cancer and stem cells.

MYC Overexpression Induces Prostatic Intraepithelial Neoplasia and Loss of Nkx3.1 in Mouse Luminal Epithelial Cells

Lo-MYC and Hi-MYC mice develop prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate[1]. However, prior studies have not determined precisely when, and in which cell types, MYC is induced. Using immunohistochemistry (IHC) to localize MYC expression in Lo-MYC transgenic mice, we show that morphological and molecular alterations characteristic of high grade PIN arise in luminal epithelial cells as soon as MYC overexpression is detected. These changes include increased nuclear and nucleolar size and large scale chromatin remodeling. Mouse PIN cells retained a columnar architecture and abundant cytoplasm and appeared as either a single layer of neoplastic cells or as pseudo-stratified/multilayered structures with open glandular lumina—features highly analogous to human high grade PIN. Also using IHC, we show that the onset of MYC overexpression and PIN development coincided precisely with decreased expression of the homeodomain transcription factor and tumor suppressor, Nkx3.1. Virtually all normal appearing prostate luminal cells expressed high levels of Nkx3.1, but all cells expressing MYC in PIN lesions showed marked reductions in Nkx3.1, implicating MYC as a key factor that represses Nkx3.1 in PIN lesions. To determine the effects of less pronounced overexpression of MYC we generated a new line of mice expressing MYC in the prostate under the transcriptional control of the mouse Nkx3.1 control region. These “Super-Lo-MYC” mice also developed PIN, albeit a less aggressive form. We also identified a histologically defined intermediate step in the progression of mouse PIN into invasive adenocarcinoma. These lesions are characterized by a loss of cell polarity, multi-layering, and cribriform formation, and by a “paradoxical” increase in Nkx3.1 protein. Similar histopathological changes occurred in Hi-MYC mice, albeit with accelerated kinetics. Our results using IHC provide novel insights that support the contention that MYC overexpression is sufficient to transform prostate luminal epithelial cells into PIN cells in vivo. We also identified a novel histopathologically identifiable intermediate step prior to invasion that should facilitate studies of molecular pathway alterations occurring during early progression of prostatic adenocarcinomas.

Alterations in nucleolar structure and gene expression programs in prostatic neoplasia are driven by the MYC oncogene

Increased nucleolar size and number are hallmark features of many cancers. In prostate cancer, nucleolar enlargement and increased numbers are some of the earliest morphological changes associated with development of premalignant prostate intraepithelial neoplasia (PIN) lesions and invasive adenocarcinomas. However, the molecular mechanisms that induce nucleolar alterations in PIN and prostate cancer remain largely unknown. We verify that activation of the MYC oncogene, which is overexpressed in most human PIN and prostatic adenocarcinomas, leads to formation of enlarged nucleoli and increased nucleolar number in prostate luminal epithelial cells in vivo. In prostate cancer cells in vitro, MYC expression is needed for maintenance of nucleolar number, and a nucleolar program of gene expression. To begin to decipher the functional relevance of this transcriptional program in prostate cancer, we examined FBL (encoding fibrillarin), a MYC target gene, and report that fibrillarin is required for proliferation, clonogenic survival, and proper ribosomal RNA accumulation/processing in human prostate cancer cells. Further, fibrillarin is overexpressed in PIN lesions induced by MYC overexpression in the mouse prostate, and in human clinical prostate adenocarcinoma and PIN lesions, where its expression correlates with MYC levels. These studies demonstrate that overexpression of the MYC oncogene increases nucleolar number and size and a nucleolar program of gene expression in prostate epithelial cells, thus providing a molecular mechanism responsible for hallmark nucleolar alterations in prostatic neoplasia.

Molecular Alterations in Prostate Cancer as Diagnostic, Prognostic, and Therapeutic Targets

Prostatic adenocarcinoma is extremely common in Western nations, representing the second leading cause of cancer death in American men. The recent application of increasingly sophisticated molecular approaches to the study of prostate cancer in this “postgenomic” era has resulted in a rapid increase in the identification of somatic genome alterations and germline heritable risk factors in this disease. These findings are leading to a new understanding of the pathogenesis of prostate cancer and to the generation of new targets for diagnosis, prognosis, and prediction of therapeutic response. Although we are still in the very early phase of clinical development, some of the molecular alterations identified in prostate cancer are being translated into clinical practice. The purpose of this review is to update the practicing surgical pathologist, and residents-in-training in pathology, regarding recent findings in the molecular pathobiology of prostate cancer. We will highlight some of the somatic molecular alterations associated with prostate cancer development and progression, with a focus on newer discoveries. In addition, recent studies in which new molecular diagnostic approaches have been applied in the clinic will be discussed.

Treatment with riboflavin and ultraviolet light prevents alloimmunization to platelet transfusions and cardiac transplants

Background. Functional leukocytes in blood transfusions can cause alloimmunization. Previous studies have shown that exposure of platelet concentrates to riboflavin and light (Mirasol PRT treatment) causes irreparable modification of nucleic acids. This treatment does not interfere with platelet function but does inhibit a wide range of immunological functions of leukocytes present in platelet concentrates. The current study evaluated the ability of Mirasol treatment to prevent alloimmunization by platelet transfusions in rats. Methods. Lewis rats received eight transfusions of untreated or Mirasol-treated platelets containing leukocytes from DA rats. Animals were subsequently challenged with a heart transplant under cyclosporine treatment. Results. Mirasol treatment caused apoptosis of the leukocytes as measured by annexin V and CD45 staining. Complement split products were deposited on the apoptotic bodies in the platelet pack. The primary and secondary immunoglobulin (Ig) M and IgG responses in rats that received Mirasol-treated platelets were almost completely abolished compared to animals that received untreated platelets. Untreated platelet transfusions elicited strong IgG responses that were associated with rapid rejection of subsequent heart transplants. Rejected hearts contained macrophage infiltrates and C4d deposits. In contrast, no grafts were rejected by recipients transfused with Mirasol-treated platelets. Macrophage infiltrates and C4d deposits were decreased in these grafts. Recipients that were presensitized to untreated platelets were capable of producing a memory response to Mirasol-treated platelets that caused accelerated rejection of subsequent transplants. Conclusions. Transfusions of platelets that were treated with riboflavin and ultraviolet light prevented presensitization to transplants. However, Mirasol-treated platelets were immunogenic in presensitized recipients.

Overexpression of ribosomal RNA in prostate cancer is common but not linked to rDNA promoter hypomethylation

Alterations in nucleoli, including increased numbers, increased size, altered architecture and increased function are hallmarks of prostate cancer cells. The mechanisms that result in increased nucleolar size, number and function in prostate cancer have not been fully elucidated. The nucleolus is formed around repeats of a transcriptional unit encoding a 45S ribosomal RNA (rRNA) precursor that is then processed to yield the mature 18S, 5.8S and 28S RNA species. Although it has been generally accepted that tumor cells overexpress rRNA species, this has not been examined in clinical prostate cancer. We find that indeed levels of the 45S rRNA, 28S, 18S and 5.8S are overexpressed in the majority of human primary prostate cancer specimens as compared with matched benign tissues. One mechanism that can alter nucleolar function and structure in cancer cells is hypomethylation of CpG dinucleotides of the upstream rDNA promoter region. However, this mechanism has not been examined in prostate cancer. To determine whether rRNA overexpression could be explained by hypomethylation of these CpG sites, we also evaluated the DNA methylation status of the rDNA promoter in prostate cancer cell lines and the clinical specimens. Bisulfite sequencing of genomic DNA revealed two roughly equal populations of loci in cell lines consisting of those that contained densely methylated deoxycytidine residues within CpGs and those that were largely unmethylated. All clinical specimens also contained two populations with no marked changes in methylation of this region in cancer as compared with normal. We recently reported that MYC can regulate rRNA levels in human prostate cancer; here we show that MYC mRNA levels are correlated with 45S, 18S and 5.8S rRNA levels. Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels. We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells. Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression.

Storage of Bacteria and Yeast

Yeast and bacteria can be cryopreserved and stored almost indefinitely. It is useful to prepare stocks for archival purposes.