Menin acts as contextual a tumor suppressor and a tumor promoter, partly via epigenetic regulation of gene transcription. While menin is phosphorylated, it remains unclear whether wild type menin has other post-translational modifications. Here, we report that menin is SUMOylated by SUMO1 in vivo and in vitro, and the SUMOylation is reduced by a SUMO protease. Lysine 591 of menin was covalently modified by SUMO1 and K591R mutation in menin blocked SUMOylation of the C-terminal part of menin in transfected cells. Full-length menin with K591 mutation was still SUMOylated in vivo, suggesting the existence of multiple SUMOylation sites. Menin K591R mutant or menin-SUMO fusion protein still retains the ability to regulate cell proliferation and the expression of the examined menin target genes.
Menin; SUMOylation; SUMO1; K591R
Clear cell carcinoma of the endometrium (CCC) is an uncommon histotype whose analyses have generally been hampered by its rarity and issues of interobserver diagnostic variability. In this study, we analyzed the clinicopathologic features of 50 CCCs that were assembled from multiple institutions and which we considered to be morphologically unambiguous after a rigorous review process for diagnostic accuracy. Forty-four (88%) of the 50 CCC cases showed an admixture of the classic architectural patterns (glandular, papillary, solid and cystic in decreasing order of prevalence). Mitotic indices were variable but were generally low: 60% of cases had a mitotic index of 3 or lower. The predominant cell type lining glands and papillae was invariably hobnail and/or cuboidal. Stratification of nuclei (greater than 3 cells) or columnar cells on glands and papillae were uncommon and never diffusely present. 82% of cases showed an admixture of polygonal cells with clear and eosinophilic cytoplasm; only clear cells were present in 4% and only eosinophilic cells were present in 10%. Hobnail cells were common, being identifiable in 86% of cases, and being diffuse in 60%. Only 2 cases had a predominance of nuclear grade 3 cells. Psammoma, hyaline and targetoid bodies were identified in 32%, 52% and 20% of cases respectively. Clear cell endometrial intraepithelial carcinoma was identified in 41.7% of cases with evaluable background endometrium. The 5-year progression free survival (PFS) for the entire cohort was 61%, and was 88%, 75%, 22% and 28.6% for stages I to IV respectively. On univariate analyses, age >65 years, advanced FIGO stage, and the presence of any lymph node metastases were associated with reduced PFS (p=0.02, 0.002, and 0.002 respectively). On multivariate analyses, the only variable associated with reduced PFS was age >65 years. The 5-year overall survival (OS) for the entire cohort was 78%, and was 94%, 87.5%, 66.7%, and 42.8% for stages I to IV respectively. On univariate analyses, the following factors were associated with reduced OS: age >65 years (p=0.04), advanced FIGO stage (p=0.003), distant metastases (p=0.003), myometrial invasion >30% (p=0.01), a mitotic index >4 (p=0.014), and a specific architectural pattern (at least 10% of the tumor composed of solid masses or individual infiltrating tumor cells, p=0.02). On multivariate analyses, only age >65 years and advanced stage were associated with reduced OS (p=0.023 and 0.022 respectively). In summary, endometrial CCC has a wide morphologic spectrum that is detailed and illustrated herein, but also has core cytoarchitectural features that are of high diagnostic utility. Morphologically unambiguous CCC apparently have patient outcomes that are more favorable than has previously been reported, indicating that ambiguous tumors should be classified separately. The existence of morphologically ambiguous clear-cell rich carcinomas that do not fit the conventional histotypic groupings, is a likely reflection of the biologic complexity of endometrial carcinomas in general; these cases should be reported descriptively, and studied separately from conventional CCC.
Clear cell carcinoma; endometrium; morphologic features
Cholesterol is a fundamental molecule for life. Located in the cell membrane, this sterol participates to the cell signaling of growth factors. Inside the cell it can be converted in hormones such as androgens or modulate the immune response. Such important functions could not be solely dependent of external supply by diet hence de novo synthesis could occur from acetate in almost all mammalian cells. If a deficiency in cholesterol sourcing leads to development troubles, overstocking has been associated to various diseases such as atherosclerosis and cancers. Cholesterol homeostasis should thus be tightly regulated at the uptake, de novo synthesis, storage and export processes. Various transcription factors have been described these last years as important to regulate cholesterol levels. Besides, synthetic molecules have been developed for many years to modulate cholesterol synthesis, such as statins. Many articles have associated prostate cancer, whose incidence is constantly increasing, to cholesterol disequilibrium. Targeting cholesterol could thus be a new pharmacological hit to counteract the initiation, development and/or progression of prostate cancer. Among the transcription factors regulating cholesterol homeostasis, the nuclear receptors Liver X Receptors (LXRs) control cholesterol uptake and export. Targeting the LXRs offers a new field of investigation to treat cancer. This review highlights the molecular relationships among LXRs, prostate cancer and cholesterol and why LXRs have good chance to be targeted one day in this tumor. LXRs, prostate cancer and cholesterol, more than a “Ménage à trois”, The Good, the Bad and the Ugly.
LXR; cholesterol; prostate cancer; lipid raft; pharmacological modulation
Endosulfatases HSulf-1 and -2 (also referred to as Sulf1 and -2) represent a family of enzymes that modulate heparin binding growth factor signaling. Heparan sulfatase 1 (HSulf-1) and heparan sulfatase 2 (HSulf-2) are two important 6-O endosulfatases which remove or edit 6-O sulfate residues of N-glucosamine present on highly sulfated HS. Alteration of heparan sulfatases have been identified in the context of several cancer types. Many cancer types either exhibit increased or decreased HSulfs expression at the transcript levels. Specifically, HSulf-1 was found to be downregulated in early-stage ovarian tumors, hepatocellular carcinoma, and metastatic breast cancer patients. HSulf-2 was found to be upregulated in ductal carcinoma in situ and invasive ductal carcinoma, whereas limited information is present about HSulf-2 expression in different stages of ovarian cancers. Here, we review the important role of these sulfatases play in ovarian and breast cancers in terms of tumorigenesis such as angiogenesis, chemoresistance, apoptosis, growth factor signaling, hypoxia and metastasis. These recent discoveries have added significant understanding about these sulfate editing enzymes.
Ovarian and breast cancer; heparin binding growth factor signaling; tumorigenesis; angiogenesis; chemoresponse and metastasis
Despite remaining uncertainties and ongoing research it is possible to draw up a model for the role of (cancer) stem cells in both the initiation and progression of cancer towards metastasis. The cancer stem cell of origin and the cancer stem cell are, despite phenotypic similarities, genotypically different entities. Given the right circumstances provided by a combination of genomic changes and biochemical and physical interactions with its microenvironment, an epithelial cancer cell may undergo a phenotypic epithelial mesenchymal transition (EMT) towards a cancer stem cell. This transition conveys upon the cell crucial stem cell-like abilities which facilitate migration into the blood circulation as an individual circulating tumor cell, survive there, and subsequently seed into organ tissue where, once more in close interaction with its microenvironment, the process of clonal self renewal may start, leading to a metastatic tumor. Both in the primary tumor as well as in the metastatic tumor, partial differentiation of the cancer stem cell progeny leads to phenotypic heterogeneity. Throughout this complex process of cancer metastasis similarities with the way stem cells function during embryonic development, including the signaling pathways that mediate these functions, are evident. Deeper insight in the EMT process, plasticity of the resulting cancer stem cells, and the role of cancer stem cells in the metastatic process is expected to lead to novel anti-metastatic cancer therapies. Emerging human in vitro cancer models in the form of “organ-on-a-chip” may contribute valuable novel research tools to achieve this aim.
Cancer stem cell Wnt signaling pathway circulating tumor evolution metastasis
Breast cancer is one of the leading causes of cancer related deaths worldwide. Breast cancer-related mortality is associated with the development of metastatic potential of primary tumor lesions. The chemokine receptor CXCR4 has been found to be a prognostic marker in various types of cancer, including breast cancer. Recent advances in the field of cancer biology has pointed to the critical role that CXCR4 receptor and its ligand CXCL12 play in the metastasis of various types of cancer, including breast cancer. Breast tumors preferentially metastasize to the lung, bones and lymph nodes, all of which represent organs that secrete high levels of CXCL12. CXCL12 acts as a chemoattractant that drives CXCR4-positive primary tumor cells towards secondary metastatic sites leading to the onset of metastatic lesions. Since its discovery in 2001, the CXCR4 field has progressed at a very fast rate and further studies have pointed to the role of CXCR4 in dissemination of tumor cells from primary sites, transendothelial migration of tumor cells as well as the trafficking and homing of cancer stem cells. This review summarizes the information that has been obtained over the years regarding the role of CXCL12-CXCR4 signaling in breast cancer, discusses its potential application to the development of new therapeutic tools for breast cancer control, and elucidates the potential therapeutic challenges which lie ahead and the future directions that this field can take for the improvement of prognosis in breast cancer patients.
CXCR4; CXCL12; breast cancer
High mobility group box 1 (HMGB1), an evolutionarily highly conserved and abundant nuclear protein also has roles within the cytoplasm and as an extracellular damage-associated molecular pattern (DAMP) molecule. Extracellular HMGB1 is the prototypic endogenous ‘danger signal’ that triggers inflammation and immunity. Recent findings suggest that posttranslational modifications dictate the cellular localization and secretion of HMGB1. HMGB1 is actively secreted from immune cells and stressed cancer cells, or passively released from necrotic cells. During cancer development or administration of therapeutic agents including chemotherapy, radiation, epigenetic drugs, oncolytic viruses, or immunotherapy, the released HMGB1 may either promote or limit cancer growth, depending on the state of progression and vascularization of the tumor. Extracellular HMGB1 enhances autophagy and promotes persistence of surviving cancer cells following initial activation. When oxidized, it chronically suppresses the immune system to promote cancer growth and progression, thereby enhancing resistance to cancer therapeutics. In its reduced form, it can facilitate and elicit innate and adaptive anti-tumor immunity, recruiting and activating immune cells, in conjunction with cytotoxic agents, particularly in early transplantable tumor models. We hypothesize that HMGB1 also functions as an epigenetic modifier, mainly through regulation of NF-kB-dependent signaling pathways, to modulate the behavior of surviving cancer cells as well as the immune cells found within the tumor microenvironment. This has significant implications for developing novel cancer therapeutics.
Cancer; HMGB1; NF-kB signaling; activation; innate immunity; dendritic cells; CD8+ T cells; epigenetic pathways
During carcinogenesis, tumors induce dysfunctional development of hematopoietic cells. Myeloid lineage cells, in the form of myeloid derived suppressor cells (MDSCs) and alternatively polarized M2 macrophages, influence almost all types of cancers by regulating diverse facets of immunosuppression, angiogenesis, cell proliferation, growth and metastasis. One-third of Americans are obese, and accumulating evidence suggests that obesity is a risk factor for various cancers. However, the relationship between these immune players and obesity are not well-described. In this review, we evaluate potential mechanisms through which different aspects of obesity, namely insulin resistance, increased estrogen, adiposity and low grade chronic inflammation from adipose tissue macrophages, may coalesce to promote MDSC induction and M2 macrophage polarization, thereby facilitating cancer development. Detailed understanding of the interplay between obesity and myeloid mediated immunosuppression may provide novel avenues for therapeutic targeting, with the goal to reduce the challenge obesity presents towards gains made in cancer outcomes.
Obesity; inflammation; myeloid derived suppressor cells; alternately activated macrophage; cancer
FDA approval of new therapies in 2011 has greatly expanded the treatment options for metastatic melanoma. Patients with V600 mutant v-raf murine sarcoma viral oncogene homolog B1 (B-RAF) positive metastatic melanoma are now treated with the RAF inhibitor, vemurafenib (Zelboraf) as a first line therapy. Vemurafenib decreases tumor size by at least 30% in approximately 50% of patients and increases progression-free survival and overall patient survival compared to the previous standard-of-care, dacarbazine. However, some patients treated with vemurafenib fail to show significant tumor shrinkage, and most patients who initially respond to the drug eventually show disease progression. Therefore, there is a clinical need to improve efficacy and prevent resistance to vemurafenib. It has been previously shown that cell death resulting from RAF/mitogen-activated protein kinase kinase (MEK) inhibition is largely dependent on increased expression of pro-apoptotic, Bcl-2 homology domain (BH3)-only proteins, such as Bcl-2-like 11 (Bim-EL) and Bcl-2 modifying factor (Bmf). Here, we show that contrary to expression of Bim-EL and Bmf, the pro-apoptotic, BH3-only protein, phorbol-12-myristate-13-acetate-induced protein 1 (Noxa), is strongly downregulated after RAF/MEK inhibition. This downregulation occurs at both the protein and mRNA level of expression and is associated with the inhibition of cell cycle progression. Restoring expression of Noxa in combination with RAF/MEK inhibition enhances cell death. Co-expression of the pro-survival, B-cell CLL/lymphoma 2 (Bcl-2) family member, myeloid cell leukemia sequence 1 (Mcl-1), with Noxa fully mitigates the enhanced cell death associated with increased Noxa expression. These data indicate that manipulating the Noxa/Mcl-1 axis may enhance the efficacy of RAF/MEK inhibitors.
Melanoma; B-RAF; Noxa; RAF/MEK inhibition
Therapeutic monoclonal antibodies (mAbs) that target the CD20 antigen on B cells are successfully used in the clinic for the depletion of B cells to treat various forms of cancer and autoimmune diseases. The first CD20 mAb, approved by the FDA in 1998, was rituximab (RTX) and since then it has been widely used to treat more than one million patients thus far. The success of RTX has led to a general interest in the mechanism of action of CD20 mAbs. CD20 mAbs can induce tumor killing via various mechanisms, such as direct induction of apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent lysis (CDC). Although we now understand these mechanisms better, it is still unclear which of these mechanisms is the most important for in vivo RTX action. Not every patient respond to RTX treatment and eventually the overwhelming majority will experience a relapse. Therefore, there is an urgent need to improve the efficacy of CD20 mAbs. This review aims to summarize our current understanding on the mechanism of action of CD20 mAbs.
Antibodies; CD20; effector mechanisms; Fc receptors; complement; complement receptors; apoptosis
Recent technological advancements in gene expression analysis have led to the discovery of a promising new group of prostate cancer (PCa) biomarkers that have the potential to influence diagnosis and the prediction of disease severity. The accumulation of deleterious changes in gene expression is a fundamental mechanism of prostate carcinogenesis. Aberrant gene expression can arise from changes in epigenetic regulation or mutation in the genome affecting either key regulatory elements or gene sequences themselves. At the epigenetic level, a myriad of abnormal histone modifications and changes in DNA methylation are found in PCa patients. In addition, many mutations in the genome have been associated with higher PCa risk. Finally, over- or underexpression of key genes involved in cell cycle regulation, apoptosis, cell adhesion and regulation of transcription has been observed. An interesting group of biomarkers are emerging from these studies which may prove more predictive than the standard prostate specific antigen (PSA) serum test. In this review, we discuss recent results in the field of gene expression analysis in PCa including the most promising biomarkers in the areas of epigenetics, genomics and the transcriptome, some of which are currently under investigation as clinical tests for early detection and better prognostic prediction of PCa.
Prostate cancer; biomarker; epigenetics; methylation; acetylation; ncRNA; genomics; SNP; transcriptomics; miRNA; lncRNA
There are over 150 human proteins that have been categorized as bona fide DNA repair proteins. These DNA repair proteins maintain the integrity of the genome, reducing the onset of cancer, disease and aging phenotypes. Variations in expression and/or function would therefore impact genome integrity as well as the cellular response to genotoxins. Global gene expression analysis is an effective approach to uncover defects in DNA repair gene expression and to discover cellular and/or organismal effects brought about by external stimuli such as environmental genotoxicants, chemotherapeutic regimens, viral infections as well as developmental and age-related stimuli. Given the significance of genome stability in cell survival and response to stimuli, we have hypothesized that cells may undergo transcriptional re-programming to accommodate defects in basal DNA repair capacity to promote survival. As a test of this hypothesis, we have compared the transcriptome in three DNA polymerase ß knockout (Polß-KO) mouse embryonic fibroblasts (MEFs) and the corresponding wild-type (WT) littermate control cell lines. Each Polß-KO cell line was found to have a range of genes up-regulated, when compared to its WT littermate control cell line. Interestingly, six (6) genes were commonly up regulated in all three Polß-KO cell lines, including Sox2, one of several genes associated with the induction of pluripotent stem cells. Herein, we present these findings and suggest that loss of DNA repair and the induction of cellular transcriptional re-programming may, in part, contribute to tumor formation and the cellular response to external stimuli.
DNA polymerase ß; mouse embryonic fibroblast; Sox2; gene expression profiling; transcriptional reprogramming
The c-Jun N-terminal Kinases (JNK), along with Erk and p38, constitute the principle members of the mitogen-activated protein kinase (MAPK) family. JNK functions primarily through AP1 family transcription factors to regulate a plethora of cellular processes, including cell proliferation, differentiation, survival and migration. It also cross-talks and integrates with other signaling pathways in a cell context-specific and cell type-specific manner. The current views of JNK function in various skin cancers and the need of developing JNK subunit-specific inhibitors for cancer type-specific applications have been summarized in this review.
JNK; skin cancer; squamous cell carcinoma; basal cell carcinoma; cylindroma
The Drosophila DNA replication-related element-binding factor (dDREF) has been identified as a master regulator of cell proliferation-related genes via its binding to the DRE sequence, 5′-TATCGATA. However, the biological roles of DREF are still to be clarified. Here, we show that DREF mutant females have steroid hormone ecdysone-deficient phenotypes, such as the loss of vitellogenic egg chambers. Furthermore, DREF knockdown in the prothoracic gland of larva prevented pupation and this was rescued via 20-hydroxyecdysone treatment. We found a DRE-like sequence (-625 to -632) in the 5′-flanking region of the Drosophila shadow gene, which catalyzes the conversion of 2-deoxyecdysone to ecdysone, and demonstrated that shadow is a novel target gene of dDREF using quantitative RT-PCR and Chip assays. In addition, we show that the level of dDREF protein correlated with age-related changes in the level of shadow mRNA in the ovaries of wild-type flies. Taken together, our data indicate that dDREF plays a key role in steroid synthesis via regulation of the shadow gene.
Drosophila; DRE; DREF; transcriptional regulation; ecdysone; shadow; steroidogenesis
We have previously reported genetic differences between Western and Chinese prostate cancers, including different frequencies of ERG rearrangements. We investigated further ERG expression and rearrangements in prostate cancers and high-grade prostatic intraepithelial neoplasia (HGPIN) from the UK and China to determine differences between these two populations by tissue microarray based immunohistochemistry and fluorescence in situ hybridization. In keeping with our previous observation, that ERG was rearranged at a higher frequency in UK prostate cancer samples (38%, 58/155) than Chinese ones (8%, 7/93), ERG rearrangements were also found in 21% (4/19) and 0% (0/19) foci of HGPIN in UK and Chinese samples respectively. ERG nuclear expression in UK cancers (34%, 54/160) was significantly higher than that in Chinese ones (10%, 9/88) (p<0.001). ERG nuclear expression in UK HGPIN (28%, 11/39) was higher than that in Chinese HGPIN (0%, 0/9), but without statistical significance (p=0.193). ERG nuclear expression was correlated to ERG rearrangements in both UK (Kappa=0.686) and Chinese (Kappa=0.565) cancers. These data demonstrate that ERG rearrangement and expression frequencies are different in prostate cancers from UK and China as early as the precursor lesion, HGPIN. The nuclear expression is associated with ERG rearrangements which mainly occur in the Western samples. UK and Chinese prostate cancers may be the result of different genetic mechanisms.
ERG; prostate cancer; high-grade prostatic intraepithelial neoplasia; genomic rearrangement; protein expression
Genome-wide studies have revealed that human and other mammalian genomes are pervasively transcribed and produce thousands of regulatory non-protein-coding RNAs (ncRNAs), including miRNAs, siRNAs, piRNAs and long non-coding RNAs (lncRNAs). Emerging evidences suggest that these ncRNAs also play a pivotal role in genome integrity and stability via the regulation of DNA damage response (DDR). In this review, we discuss the recent finding on the interplay of ncRNAs with the canonical DDR signaling pathway, with a particular emphasis on miRNAs and lncRNAs. While the expression of ncRNAs is regulated in the DDR, the DDR is also subjected to regulation by those DNA damage-responsive ncRNAs. In addition, the roles of those Dicer- and Drosha-dependent small RNAs produced in the vicinity of double-strand breaks sites are also described.
DNA damage response; ncRNAs; miRNAs; lncRNAs; crosstalk
The development of new cancer therapeutics would benefit from incorporating efficient tumor models that mimic human disease. We have developed a subcutaneous bladder tumor regeneration system that recapitulates primary human bladder tumor architecture by recombining benign human fetal bladder stromal cells with SW780 bladder carcinoma cells. As a first step, SW780 cells were seeded in ultra low attachment cultures in order to select for sphere-forming cells, the putative cancer stem cell (CSC) phenotype. Spheroids were combined with primary human fetal stromal cells or vehicle control and injected subcutaneously with Matrigel into NSG mice. SW780 bladder tumors that formed in the presence of stroma showed accelerated growth, muscle invasion, epithelial to mesenchymal transition (EMT), decreased differentiation, and greater activation of growth pathways compared to tumors formed in the absence of fetal stroma. Tumors grown with stroma also demonstrated a greater similarity to typical malignant bladder architecture, including the formation of papillary structures. In an effort to determine if cancer cells from primary tumors could form similar structures in vivo using this recombinatorial approach, putative CSCs, sorted based on the CD44+CD49f+ antigenic profile, were collected and recombined with fetal bladder stromal cells and Matrigel prior to subcutaneous implantation. Retrieved grafts contained tumors that exhibited the same structure as the original primary human tumor. Primary bladder tumor regeneration using human fetal bladder stroma may help elucidate the influences of stroma on tumor growth and development, as well as provide an efficient and accessible system for therapeutic testing.
Bladder cancer; cancer stem cell (CSC); subcutaneous tumor model; stroma; sphere
Accumulated evidence suggests that thyroid hormone receptor β (TRβ) could function as a tumor suppressor, but the detailed mechanisms by which TRβ inhibits tumorigenesis are not fully understood. The present studies explored the mechanisms by which TRβ acted to inhibit thyroid tumor development mediated by simian virus-40 (SV40). In mouse xenograft models, SV40 large T antigen (SV40Tag)-immortalized human thyroid epithelial (HTori) cells rapidly induced tumors, but the tumor development was totally blocked by TRβ stably expressed in HTori cells. Previous studies showed that the SV40Tag oncoprotein binds to and inactivates tumor suppressors p53 and retinoblastoma protein (Rb), thereby inducing tumorigenesis. Here we showed that one of the mechanisms by which TRβ suppressed tumor development was by competing with p53 and Rb for binding to SV40Tag. The interaction of TRβ with SV40Tag led to reactivation of Rb to inhibit cell cycle progression. TRβ- SV40Tag interaction also resulted in reactivating p53 to increase the expression of Pten, thus attenuating PI3K-AKT signaling to decrease cell proliferation and to induce apoptosis. The present study uncovered a novel action of TRβ as a tumor suppressor initiated via interfering with the recruitment of Rb and p53 by SV40Tag oncoprotein through protein-protein interaction, thereby acting to block tumor development.
Thyroid hormone receptor; tumor suppressor; tumorigenesis; thyroid hormone; xenograft models
Our purpose is to develop a serum assay to determine an individual’s probability of having colorectal cancer (CRC). We have discovered a protein panel yielding encouraging, clinically significant results. We evaluated 431 serum samples from donors screened for CRC by colonoscopy. We compared the concentration of seven proteins in individuals with CRC versus individuals found to be CRC free. The assay monitored a single peptide from each of seven proteins. Comparing CRC to normal samples in univariate two-sample t-tests, 6 of the 7 proteins yielded a p-value less than 0.01. Logistic regression was used to construct a model for determination of CRC probability. The model was fit on a randomly chosen training set of 321 samples. Using 6 of the 7 proteins (ORM1, GSN, C9, HABP2, SAA2, and C3) and a cut point of 0.4, an independent test set of 110 samples yielded a sensitivity of 93.75%, a specificity of 82.89% and a prevalence-adjusted negative predictive value (NPV) of 99.9775% for the assay. The results demonstrate that the assay has promise as a sensitive, non-invasive diagnostic test to provide individuals with an understanding of their own probability of having CRC.
Colon cancer; proteomics; cancer; colon; mass spec; MRM; colorectal; CRC
The p53 tumor suppressor pathway is inactivated in cancer either via direct mutation or via deregulation of upstream regulators or downstream effectors. P53 mutations are rare in uveal melanoma. Here we investigated the role of the p53 inhibitor Hdmx in uveal melanoma. We found Hdmx over-expression in a subset of uveal melanoma cell lines and fresh-frozen tumor samples. Hdmx depletion resulted in cell-line dependent growth inhibition, apparently correlating with differential Hdm2 levels. Surprisingly, p53 knockdown hardly rescued cell cycle arrest and apoptosis induction upon Hdmx knockdown, whereas it effectively prevented growth suppression induced by the potent p53 activator Nutlin-3. In addition, two compounds inhibiting Hdmx function or expression, SAH-p53-8 and XI-011, also elicited a growth inhibitory effect in a partly p53-independent manner. These findings suggest a novel, growth-promoting function of Hdmx that does not rely on its ability to inhibit p53. We provide evidence for a contribution of p27 protein induction to the observed p53-independent G1 arrest in response to Hdmx knockdown. In conclusion, our study establishes the importance of Hdmx as an oncogene in a subset of uveal melanomas and widens the spectrum of its function beyond p53 inhibition.
Uveal melanoma; Hdmx; p53; Nutlin-3; p27; SAH-p53-8; XI-011; retinoblastoma
Acute myeloid leukemia (AML) is the result of a multistep transforming process of hematopoietic precursor cells (HPCs) which enables them to proceed through limitless numbers of cell cycles and to become resistant to cell death. Increased proliferation renders these cells vulnerable to acquiring mutations and may favor leukemic transformation. Here, we review how deregulated cell cycle control contributes to increased proliferation in AML and favors genomic instability, a prerequisite to confer selective advantages to particular clones in order to adapt and independently proliferate in the presence of a changing microenvironment. We discuss the connection between differentiation and proliferation with regard to leukemogenesis and outline the impact of specific alterations on response to therapy. Finally, we present examples, how a better understanding of cell cycle regulation and deregulation has already led to new promising therapeutic strategies.
Acute myeloid leukemia (AML); cell cycle; genetic instability; proliferation; differentiation
The behavior of tumor cells is influenced by the composition of the surrounding tumor environment. The importance of ascites in ovarian cancer (OC) progression is being increasingly recognized. The characterization of soluble factors in ascites is essential to understand how this environment affects OC progression. The development of cytokine arrays now allows simultaneous measurement of multiple cytokines per ascites using a single array.
We applied a multiplex cytokine array technology that simultaneously measures the level of 120 cytokines in ascites from 10 OC patients. The ascites concentration of a subset (n = 5) of cytokines that was elevated based on the multiplex array was validated by commercially available ELISA. The ascites level of these 5 cytokines was further evaluated by ELISA in a cohort of 38 patients. Kaplan-Meier analysis was used to assess the association of cytokine expression with progression-free survival (PFS) in this cohort.
We observed a wide variability of expression between different cytokines and levels of specific cytokines also varied in the 10 malignant ascites tested. Fifty-three (44%) cytokines were not detected in any of the 10 ascites. The level of several factors including, among others, angiogenin, angiopoietin-2, GRO, ICAM-1, IL-6, IL-6R, IL-8, IL-10, leptin, MCP-1, MIF NAP-2, osteprotegerin (OPG), RANTES, TIMP-2 and UPAR were elevated in most malignant ascites. Higher levels of OPG, IL-10 and leptin in OC ascites were associated with shorter PFS. IL-10 was shown to promote the anti-apoptotic activity of malignant ascites whereas OPG did not.
Our data demonstrated that there is a complex network of cytokine expression in OC ascites. Characterization of cytokine profiles in malignant ascites may provide information from which to prioritize key functional cytokines and understand the mechanism by which they alter tumor cells behavior. A better understanding of the cytokine network is essential to determine the role of ascites in OC progression.
Ascites; ovarian cancer; tumor environment; cytokines; mulitplex array; IL-10
RanBPM is a ubiquitous protein that has been reported to regulate several cellular processes through interactions with various proteins. However, it is not known whether RanBPM may regulate gene expression patterns. As it has been shown that RanBPM interacts with a number of transcription factors, we hypothesized that it may have wide ranging effects on gene expression that may explain its function. To test this hypothesis, we generated stable RanBPM shRNA cell lines to analyze the effect of RanBPM on global gene expression. Microarray analyses were conducted comparing the gene expression profile of Hela and HCT116 RanBPM shRNA cells versus control shRNA cells. We identified 167 annotated genes significantly up- or down-regulated in the two cell lines. Analysis of the gene set revealed that down-regulation of RanBPM led to gene expression changes that affect regulation of cell, tissue, and organ development and morphology, as well as biological processes implicated in tumorigenesis. Analysis of Transcription Factor Binding Sites (TFBS) present in the gene set identified several significantly over-represented transcription factors of the Forkhead, HMG, and Homeodomain families of transcription factors, which have previously been demonstrated as having important roles in development and tumorigenesis. In addition, the combined results of these analyses suggested that several signaling pathways were affected by RanBPM down-regulation, including ERK1/2, Wnt, Notch, and PI3K/Akt pathways. Lastly, analysis of selected target genes by quantitative RT-qPCR confirmed the changes revealed by microarray. Several of the genes up-regulated in RanBPM shRNA cells encode proteins with known oncogenic functions, such as the RON tyrosine kinase, the adhesion molecule L1CAM, and transcription factor ELF3/ESE-1, suggesting that RanBPM functions as a tumor suppressor to prevent deregulated expression of these genes. Altogether, these results suggest that RanBPM does indeed function to regulate many genomic events that regulate embryonic, tissue, and cellular development as well as those involved in cancer development and progression.
RanBPM; ERK; Wnt; Notch; microarray; cancer; development
Mutation in the BRCA1 gene is associated with increased risk for hereditary breast and ovarian cancers. In sporadic ovarian tumors, BRCA1 dysfunction is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein. Our group has previously reported that BRCA1 proteins, unlike K109R and cancer-predisposing mutant C61G BRCA1 proteins, bind the sole SUMO E2-conjugating enzyme Ubc9. In this study, we examined the result of altered Ubc9 binding and knockdown on the sub-cellular localization and growth inhibitory function of BRCA1 proteins in ovarian cancer cells. Using live imaging of YFP, RFP-tagged BRCA1 and BRCA1a proteins, our results show enhanced cytoplasmic localization of K109R and C61G mutant BRCA1 proteins in ES-2, NIHOVCAR3 and UWB 1.289 ovarian cancer cells. Down-regulation of Ubc9 in ovarian cancer cells using Ubc9 siRNA resulted in cytoplasmic localization of BRCA1 and BRCA1a proteins. These mutant BRCA1a proteins were impaired in their capacity to inhibit growth of ES-2 ovarian cancer cells. Several ovarian cancer cells, including a BRCA1-null ovarian cancer cell line, showed higher levels of expression of Ubc9. This is the first study demonstrating the physiological link between loss of Ubc9 binding and loss of growth suppression of disease-associated mutant BRCA1a proteins in ovarian cancer cells. BRCA1, by turning off or on Ubc9 binding, regulates growth of ovarian cancers.
BRCA1; BRCA1a; Ubc9; Ovarian cancer; RING domain mutants; nuclear import; Growth suppression
Dissemination of cancer cells is strongly associated with reduction in quality of life, worsening of prognosis, and remains the primary cause of therapeutic failure and high mortality in cancer. A crucial factor in the progression of metastases is the ability to establish a functioning blood vessel network. Consequently therapeutic strategies which selectively target tumor vasculature may hold promise for the treatment of metastatic disease. A complicating factor in the assessment of the efficacy of vascular targeting therapies is that the metastatic process can result in multiple neoplastic lesions at various stages of growth and vascularity in a single organ. The goal of this project was to utilize a rodent squamous cell carcinoma (SCCVII) model to characterize the development of metastatic lung lesions and their associated vasculature. Mice were injected with tumor cells via the tail vein to introduce a reproducible number of lung metastases. At various times after cell injection, lungs were removed and serial sections were taken throughout the lobes for morphometric analysis. Tumor volumes were calculated for each nodule using 2 hematoxylin and eosin (H&E) stained sections that were a known distance apart. Sections adjacent to those used for size determination were reserved for immunohistochemical staining with CD31 to identify blood vessels associated with each nodule. The results showed that although the median tumor volume increased from 0.006 to 0.51 mm3 between 7 and 18 days post SCCVII cell injection, a range of tumor sizes existed at all-times. Irrespective of the time of assessment, nodules with volumes ≤ 0.5 mm3 had a constant vessel density while those with volumes >0.5 mm3 showed increasing vessel densities with increasing size. These findings indicate that the methodology outlined in this study can identify metastases in various stages of vascular development and could therefore be applied to evaluate and distinguish therapeutic interventions that seek to prevent the initiation of blood vessel networks and those targeting already established expanding tumor vasculature. Examining the efficacy of such approaches, alone or in combination, in the treatment of metastases in a preclinical model could lead to the development of more effective therapeutic strategies for metastatic disease.
Metastasis; vascular development; carcinoma