Michael Karsy

Current Progress on Understanding MicroRNAs in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is an aggressive grade IV astrocytoma with a 1-year median survival rate despite current treatment modalities. A thorough understanding of the vast genetic aberrations and signaling pathways involved in gliomagenesis as well as heterogeneous clinicopathological presentation remains elusive. The recent discovery of microRNAs (miRs) and their capability of simultaneously regulating multiple downstream genes may play a key role in explaining the complex mechanisms underlying GBM formation. miRs are 19 to 25 nucleotide non-protein-coding small RNA molecules involved in the suppression of mRNA translation. This review will summarize and discuss the most recent findings regarding miRs in GBM including downstream targets, functional effects, and therapeutic potentials. Specifically discussed miRs include miR-7, miR-9/miR-9*, miR-10a/miR-10a*/miR-10b, miR-15b, miR-17-92, miR-21, miR-26a, miR-34a, miR-93, miR-101, miR-124, miR-125a, miR-125b, miR-128, miR-137, miR-146b-5p, miR-153, miR-181a/miR-181b, miR-196a/miR-196b, miR-218, miR-221/miR-222, miR-296, miR-302-367, miR-326, miR-381, miR-451, and let-7a. In addition to gene regulatory roles, miRs have demonstrated significant diagnostic, prognostic, and therapeutic potential. These small molecules may both help in the understanding of GBM and in developing new therapeutic options.

Targeting the PI3K/AKT/mTOR signaling pathway in glioblastoma: novel therapeutic agents and advances in understanding

Glioblastoma multiforme (GBM) is a grade IV astrocytoma with a median survival of 12xa0months despite current multi-modal treatment options. GBM is distinguished clinicopathologically into primary and secondary subtypes. Mutations of phosphatase and tensin homolog, and subsequent upregulation of the downstream protein kinase B/mammalian target of rapamycin (mTOR) signaling pathway, are commonly seen in primary GBM and less predominantly in secondary GBM. While investigations into targeted treatments of mTOR have been attempted, feedback regulation within the mTOR signaling pathway may account for therapeutic resistance. Currently, rapamycin analogs, dual-targeted mTOR complex 1 and 2 agents as well as dual mTOR and phosphatidylinositol-3 kinase-targeted agents are being investigated experimentally and in clinical trials. This review will discuss the experimental potential of these agents in the treatment of GBM and their current stage in the GBM drug pipeline. Knowledge obtained from the application of these agents can help in understanding the pathogenesis of GBM as well as delineating subsequent treatment strategies.

B-Raf and the inhibitors: from bench to bedside.

The B-Raf protein is a key signaling molecule in the mitogen activated protein kinase (MAPK) signaling pathway and has been implicated in the pathogenesis of a variety of cancers. An important V600E mutation has been identified and can cause constitutive B-Raf activation. Recent studies have evaluated a variety of small molecule inhibitors targeting B-Raf, including PLX4032/vemurafenib, dabrafenib, LGX818, GDC0879, XL281, ARQ736, PLX3603 (RO5212054), and RAF265. Therapeutic resistance has been identified and various mechanisms described. This review also discussed the current understanding of B-Raf signaling mechanism, methods of mutation detection, treatment strategies as well as potential methods of overcoming therapeutic resistance.

Current understanding of the role and targeting of tumor suppressor p53 in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common primary malignancy in the brain and confers a uniformly poor prognosis. Despite decades of research on the topic, limited progress has been made to improve the poor survival associated with this disease. GBM arises de novo (primary GBM) or via dedifferentiation of lower grade glioma (secondary GBM). While distinct mutations are predominant in each subtype, alterations of tumor suppressor p53 are the most common, seen in 25–30xa0% of primary GBM and 60–70xa0% of secondary GBM. Various roles of p53 that protect against neoplastic transformation include modulation of cell cycle, DNA repair, apoptosis, senescence, angiogenesis, and metabolism, resulting in an extremely complex signaling network. Mutations of p53 in GBM are most common in the DNA-binding domain, namely within six hotspot mutation sites (codons 175, 245, 248, 249, 273, and 282). These alterations generally result in loss-of-function, gain-of-function, and dominant-negative mutational effects for p53, however, the distinct effect of these mutation types in GBM pathogenesis remain unclear. Signaling alterations downstream from p53 (e.g., MDM2, MDM4, INK4/ARF), p53 isoforms (e.g., p63, p73), and microRNAs (e.g., miR-34) also play critical roles in modulating the p53 pathway. Despite novel mouse models of GBM showing that p53 combined with other mutation generate tumors de novo, the role of p53 as a molecular marker of GBM remains controversial with most studies failing to show an association with prognosis. Regarding treatment in GBM, p53 targeted-gene therapy and vaccinations have reached phase I clinical trials while therapeutic drugs are still in preclinical development. This review aims to discuss the most recent findings regarding the impact of p53 mutations on GBM pathogenesis, prognosis, and treatment.

Deciphering the signaling pathways of cancer stem cells of glioblastoma multiforme: role of Akt/mTOR and MAPK pathways.

These findings emphasize that the mTOR pathway may contribute to maintenance of quiescence of CSCs, and provide a basis for manipulating CSCs in the treatment of GBM. Future research should focus on further defining the PI3K/Akt/mTOR molecular network in the regulation of stem cell quiescence and provide rationale for targeting the cancer-initiating cells of GBM.

OC14.06: The role of antral follicle counts (AFC) via TVUSG in determining fertility preservation outcomes in cancer patients undergoing ovarian stimulation

Objectives: Chemotherapy damages ovarian reserve and induces premature ovarian failure. It has been shown that menstrual status, and cycle day 2 (CD2) FSH and estradiol (E2) levels are not reliable for the assessment of chemotherapy effects on fertility. The purpose of this study was to determine whether antral follicle count (AFC) is predictive for ovarian stimulation outcomes in pre(COH) and postchemotherapy (PCCOH) patients undergoing fertility preservation with oocyte or embryo freezing. Methods: This was a prospective study of 69 COH and 16 PCCOH women who underwent controlled ovarian hyperstimulation while AFC, anti-mullerian hormone (AMH), FSH and E2 were obtained on CD2. The predominant diagnosis was breast cancer (n = 74, 62 COH vs. 12 PCCOH). PCCOH patients received a variety of alkylating agents. Results: Total (14.4 ± 1.3 vs. 8.0 ± 1.1) and mature (9.7 ± 0.8 vs. 5.9 ± 0.9) oocytes retrieved and two pronuclear (2PN) embryos generated (7.7 ± 0.7 vs. 5.1 ± 0.8) were significantly higher for COH vs. PCCOH patients (P < 0.05). The mean age at time of stimulation, CD2-FSH, and E2 were not significantly different. AFC (13.1 ± 1.3 vs. 8.7 ± 1.2) and AMH (2.6 ± 0.4 vs. 1.1 ± 0.5) were significantly higher in the COH group (P < 0.05). Linear regression analysis for COH but not PCCOH patients demonstrated a significant positive correlation for AFC and AMH in predicting the number of total oocytes, mature oocytes and 2PN embryos (P < 0.05). Multivariate regression analysis using AFC and AMH improved prediction of these patient outcomes (R2 = 0.53). Conclusions: The results of this study demonstrated that after chemotherapy, CD2-FSH and E2 were unaltered but AMH, AFC and stimulation outcomes were significantly lower. The combined use of AFC and AMH showed a strong correlation for predicting stimulation outcomes in COH patients but further investigation is needed for PCCOH patients. Fertility preservation consultation, as well as AFC and AMH, for patients prior to chemotherapy may improve prediction of patient outcome.

Abstract 3399: STAT3 promotes metastasis to the brain: Demonstrated by protein, gene, and functional analysis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnBrain metastasis affects an estimated 10% of cancer patients with metastatic disease, a major cause of morbidity and mortality in these patients. The most common primary tumors causing brain metastasis are adenocarcinoma of the lung or breast. While recent studies have demonstrated the genetic basis of such metastases, the molecular cause(s) of brain metastases remains largely elusive. Signal transducer and activator of transcription 3 (STAT3) has been shown to control various cellular functions and regulates the immune response. STAT3 is activated in various aggressive carcinomas. We hypothesize that STAT3 may play a role in metastatic brain tumors. Metastatic brain tumors were analyzed for expression of activated STAT3Tyr705 by immunohistochemical analysis. Gene expression profiling (17,000 gene chip) was performed in metastatic tumor samples, and STAT3 regulated genes were evaluated. Functional analysis such as cell motility and proliferation were investigated by chemotactic migration and MTT assay, respectively. The results of this study showed that a significant number of metastatic tumors to brain (75%) demonstrated nuclear expression of pSTAT3Tyr705. A significant number of these tumors also expressed mutant-p53 and activated MAPK. Gene expression analysis demonstrated that 32 STAT3-associated genes were significantly altered. Upregulated genes included stathmin-1 and stathmin-like 3 that control mictotubule dynamics in migrating cells and are associated with STAT3-related migration. The upregulation of stathmin-like 3 (also called SCG10-like-protein or SCLIP) also interacts with STAT3 in the maintenance of cellular dynamics involved in cell-cell attachment. The gp130-related interleukin 6 signal transducer gene, which is generally involved in STAT3-dependent glial fibrillary acidic protein (GFAP) induction during nerve regeneration, was inhibited. The suppression of STAT3-regulated gene oncostatin M receptor, which regulates metastasis, was observed. Furthermore, protein inhibitor of activated STAT3 (PIAS3), that suppresses activated STAT3 levels, was downregulated. Functional analysis revealed that cell proliferation of breast cancer cells (MCF7 cells) was enhanced in an astrocytic environment. Chemotactic analysis showed that breast cancer cells migrated toward astrocytic media. In addition, co-culture analysis demonstrated that astrocytes and tumor cells have an affinity for each other and make genuine cell-cell contact. These results indicate that STAT3 activation plays a significant role in brain metastasis by itself as well as by altering downstream target genes that regulate various cellular functions. Furthermore, the astrocytic milieu promotes the metastatic potential of tumor cells. Therefore, the activation of STAT3 promotes metastasis to brain, and may serve as a target for therapeutic intervention.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3399.

Abstract 5153: Cancer stem cells of glioblastoma multiforme: regulation by the mTOR pathway

Glioblastoma multiforme (GBM), the most aggressive primary brain tumor, remains uniformly fatal despite current surgical, chemotherapeutic, and radiation treatment regimens. Recurrence of GBM has been associated with therapy resistant cancer stem cells (CSC), which possess the ability to generate tumors of the same genotype and phenotype as parent tumors within in vivo models. Recent evidence has suggested that neural stem and/or progenitor cells are the cell type of origin for GBM. In addition, numerous proto-oncogenes and tumor suppressor genes altered in GBM have been implicated in the self-renewal of normal stem cells, including Bmi1, Gfi1, PTEN, and p53. Aberrant PTEN/AKT signaling has been associated with the tumorigenesis of GBM. The downstream signaling target mTOR (mammalian target of Rapamycin), that exists in two distinct multicomplex proteins (mTORC1 and mTORC2), is a critical effector that is deregulated in GBM, and implies that the inhibition of mTOR may have therapeutic potential. However, inhibitors of mTOR such as Rapamycin (RAPA) or its analogs have thus far been ineffective in GBM treatment due to the activation of mitogenic pathways via feedback loops. The molecular mechanisms of CSC regulation by RAPA remain unknown. We aim to test the hypothesis that mTOR regulates CSC growth and self-renewal in GBM, and also understand the contribution of differentiation agents in targeting CSCs. For this study, we utilized primary tumor and cell line derived CSCs as evidenced by neurosphere formation. The results demonstrated that RAPA significantly suppressed neurosphere proliferation as evidenced by the inhibition of neurosphere diameter and frequency. As with mTOR inhibition, the differentiation agents arsenic trioxide (ATO), all-trans retinoic acid (ATRA), and histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) suppressed the formation of new neurospheres. Fluorescence-activated cell sorting (FACS) analysis revealed that RAPA halted GBM cells in S-phase in a time-dependent manner. In addition, a novel mTORC1 and mTORC2 inhibitor, KU0063794, significantly suppressed cell proliferation greater than RAPA alone. Also, RAPA dramatically suppressed colony formation. Prolonged mTOR inhibition with RAPA caused a shift towards mTORC2 and activation in ERK1/2 which was partially abrogated by upstream PI3K inhibition. Moreover, mTOR inhibition, but not MAPK inhibition, reduced the nuclear expression of embryonic stem cell marker Nanog. Finally, mTOR inhibition influenced cellular motility with respect to mTORC1 and mTORC2 activation. These results highlight that mTOR inhibition affects CSC proliferation and self-renewal in GBM and possibly suppresses tumorigenesis. Despite current clinical limitations, mTOR inhibitors may be effective in understanding aberrant signaling in CSCs, and aid in the design of future targeted therapies for GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5153.