Subacute neural stem cell therapy for traumatic brain injury.

INTRODUCTION: Traumatic brain injury (TBI) frequently results in devastating and prolonged morbidity. Cellular therapy is a burgeoning field of experimental treatment that has shown promise in the management of many diseases, including TBI. Previous work suggests that certain stem and progenitor cell populations migrate to sites of inflammation and improve functional outcome in rodents after neural injury. Unfortunately, recent study has revealed potential limitations of acute and intravenous stem cell therapy. We studied subacute, direct intracerebral neural stem and progenitor cell (NSC) therapy for TBI. MATERIALS AND METHODS: The NSCs were characterized by flow cytometry and placed (400,000 cells in 50 muL 1x phosphate-buffered saline) into and around the direct injury area, using stereotactic guidance, of female Sprague Dawley rats 1 wk after undergoing a controlled cortical impact injury. Immunohistochemistry was used to identify cells located in the brain at 48 h and 2 wk after administration. Motor function was assessed using the neurological severity score, foot fault, rotarod, and beam balance. Cognitive function was assessed using the Morris water maze learning paradigm. Repeated measures analysis of variance with post-hoc analysis were used to determine significance at P < 0.05. RESULTS: Immunohistochemistry analysis revealed that 1.4-1.9% of infused cells remained in the neural tissue at 48 h and 2 wk post placement. Nearly all cells were located along injection tracks at 48 h. At 2 wk some cell dispersion was apparent. Rotarod motor testing revealed significant increases in maximal speed among NSC-treated rats compared with saline controls at d 4 (36.4 versus 27.1 rpm, P < 0.05) and 5 (35.8 versus 28.9 rpm, P < 0.05). All other motor and cognitive evaluations were not significantly different compared to controls. CONCLUSIONS: Placement of NSCs led to the cells incorporating and remaining in the tissues 2 wk after placement. Motor function tests revealed improvements in the ability to run on a rotating rod; however, other motor and cognitive functions were not significantly improved by NSC therapy. Further examination of a dose response and optimization of placement strategy may improve long-term cell survival and maximize functional recovery.

DEFINING THE ROLE OF EGFR ACETYLATION IN CELLULAR PROCESSES: CLINICAL IMPLICATIONS

Epidermal growth factor receptor (EGFR) is a cell membrane tyrosine kinase receptor and plays a pivotal role in regulating cell growth, differentiation, cell cycle, and tumorigenesis. Deregulation of EGFR causes many diseases including cancers. Intensive investigation of EGFR alteration in human cancers has led to profound progress in developing drugs to target EGFR-mediated cancers. While exploring possible synergistic enhancement of therapeutic efficacy by combining EGFR tyrosine kinase inhibitors (TKI) with other anti-cancer agents, we observed that suberoylanilide hydroxamic acid (SAHA, a deacetylase inhibitor) enhanced TKI-induced cancer cell death, which further led us to question whether SAHA-mediated sensitization to TKI was associated with EGFR acetylation. What we know so far is that SAHA can inhibit class I and II histone deacetylases (HDACs), which could possibly preserve acetylation of underlying HDAC-targeted proteins including both histone and non-histone proteins. In addition, it has been reported that an HDAC inhibitor, TSA, enhanced EGFR phosphorylation in ovarian cancer cells. EGFR acetylation has also been reported to play a role in the regulation of EGFR endocytosis recently. These observations indicate that there might be an intrinsic correlation between acetylation and phosphorylation of EGFR. In other words, the interplay between EGFR acetylation and phosphorylation may contribute to HDAC inhibitors (HDACi)-augmented EGFR phosphorylation. In this investigation, we showed that CBP acetyltransferase acetylated EGFR in vivo. In response to EGF stimulation, CBP rapidly translocated from the nucleus to the cytoplasm. We also demonstrated protein-protein interaction between CBP and EGFR as well as the enhancement of EGFR acetylation by CBP. Moreover, EGFR acetylation enhanced EGFR tyrosine phosphorylation and augmented its association with Src kinase. Acetylation-deficient EGFR mutant (EGFR-K3R) significantly reduced the function and activity of EGFR. Furthermore, ectopic expression of EGFR-K3R mutant abrogated its ability to respond to EGF-induced cell proliferation, DNA synthesis, and anchorage-independent growth using cell-based assays and tumor growth in nude mice. In addition, we demonstrated that EGFR expression was associated with SAHA resistance in the treatment of cancer cells that overexpress EGFR. The knockdown of EGFR in MDA-MB-468 breast cancer cells could sensitize the cells to respond to SAHA. The overexpression of EGFR in SAHA-sensitive MDA-MB-453 breast cancer cells rendered the cells resistant to SAHA. Together, these findings suggest that EGFR plays an important role in SAHA resistance in breast carcinoma cells that we tested. The combination therapy of HDACi with TKI has been proposed for treating cancers with aberrant expression of EGFR. The evidence from pre-clinical or clinical trials demonstrated significant enhancement of therapeutic efficacy by using such a combination therapy. Our in vivo study also demonstrated that the combination of SAHA and TKI for the treatment of breast cancer significantly reduced tumor burden compared with either SAHA or TKI alone. The significance of our study elucidated another possible underlying molecular mechanism by which HDACi mediated sensitization to TKI. Our results unveiled a critical role of EGFR acetylation that regulates EGFR tyrosine phosphorylation and may further provide an experiment-based rationale for combinatorial targeted therapy.

ATM Signaling to TSC2: Mechanisms and Implications for Cancer Therapy

Ataxia telangiectasia mutated (ATM) is a critical component of the cellular response to DNA damage, where it acts as a damage sensor, and signals to a large network of proteins which execute the important tasks involved in responding to the damage, namely inducing cell cycle checkpoints, inducing DNA repair, modulating transcriptional responses, and regulating cell death pathways if the damage cannot be repaired faithfully. We have now discovered that an additional novel component of this ATM-dependent damage response involves induction of autophagy in response to oxidative stress. In contrast to DNA damage-induced ATM activation however, oxidative stress induced ATM, occurs in the cytoplasm, and does not require nuclear-to-cytoplasmic shuttling of ATM. Using several cell culture systems including MCF7 breast carcinoma cells, SKOV3 ovarian cancer cells, and various lineages of mouse embryonic fibroblasts, we showed that once activated by reactive oxygen species (ROS), ATM signals to mTORC1 to induce autophagy via the LKB1-AMPK-TSC2 pathway. Targeting dysregulation of mTORC1 in Atm-deficient mice, which succumb to lymphomagenesis within 3-4 months of age with daily administration of rapamycin, could significantly extend survival and cause regression of tumors, suggesting that pharmacologically targeting this pathway has therapeutic implications in cancer. We also identified a second contrasting pathway for DNA damage-induced mTORC1 repression which does not require AMPK activation, but does require ATM and TSC2. Several potential mechanisms including mTOR localization and p53-mediated pathways were ruled out however we identified that TSC2 may be an additional cytoplasmic direct ATM substrate that is engaged in response to DNA damage specifically. Lastly, a study was performed to examine whether autophagy induced by ovarian cancer therapeutics (focusing on cisplatin, since paclitaxel does not induce autophagy in the SKOV3 cell line model we used) plays a role in resistance to therapy since autophagy can play both pro-survival mechanisms or be a mechanism of cell death. Using a genetic approach to knock-down Atg5 expression with shRNA in SKOV3 ovarian carcinoma cells, we compared the cytotoxicity of cisplatin in vector or Atg5 knock-down cells, and demonstrated that autophagy does not play any significant role in the response to cisplatin in this cell line.

A study in the molecular and cellular functions of GSK3beta in prostate adenocarcinoma

Kinases are part of a complex network of signaling pathways that enable a cell to respond to changes in environmental conditions in a regulated and coordinated way. For example, Glycogen Synthase Kinase 3 beta (GSK3β) modulates conformational changes, protein-protein interaction, protein degradation, and activation of unique domains in proteins that transduce signals from the extracellular milieu to the nucleus. ^ In this project, I investigated the expression and function that GSK3β exhibits in prostate cells. The capacity of GSK3β to regulate two transcription factors (JUN and CREB), which are known to be inversely utilized in prostate tumor cells, was measured. JUN/AP1 is constitutively activated in PC-3 cells; whereas, CREB/CRE activity is ∼20 fold less than the former. GSK3β overexpression obliterates JUN/AP1 activity. With respect to CREB GSK3β increases CREB/CRE activity. Cellular levels of active GSK3β can determine whether JUN or CREB is preferentially active in the PC-3s. Theoretically, in response to a particular cellular context or stimulus, a cell may coordinate JUN and CREB function by regulating GSK3β.^ A comparison of various prostate cell lines showed that active GSK3β is less expressed in normal prostate epithelial cells than in tumor cells. Differentially expressed active (GSK3β) may correlate with progression of prostate carcinoma. If a known marker associated with carcinoma of the prostate could be shown to be regulated by GSK3β then, further study of GSK3β may lead to a better understanding of both possible prevention of the disease and improved therapy for advanced stages. ^ The androgen receptor (AR) is an intriguing phosphoprotein whose regulation is potentially determined by a variety of kinases. One of these is (GSK3β) I found that (GSK3β) is a regulator of the androgen receptor in both the unliganded and liganded states. It can inhibit AR function as measured by reporter assays. Also, GSK3β associates with the AR at the DNA binding domain because deletion constructs expressing either the n-terminus or the c-terminus (both having the DBD in common) immunoprecipitated with GSK3β. Increased understanding of how GSK3β functions in prostate cancer would provide clues into how (1) certain signal pathways are coordinated and (2) the androgen receptor may be regulated. ^

An interferon-inducible protein, p202, in cancer gene therapy

Interferons (IFNs) have been shown to exert antiviral, cell growth regulatory, and immunomodulatory effects on target cells. Both type I (α and β) and type II (γ) IFNs regulate cellular activities by specifically inducing the expression or activation of endogenous proteins that perform distinct biological functions. p202 is a 52 kDa nuclear phosphoprotein known to be induced by IFNs. p202 interacts with a variety of cellular transcription and growth regulatory factors and affects their functions. ^ In this report, we showed that the expression of p202 was associated with an anti-proliferative effect on human prostate cancer cells. Cells that expressed p202 showed reduced ability to grow in soft-agar, indicating a loss of transformation phenotype. More importantly, p202 expression reduced the tumorigenicity of human prostate cancer cells. p202-expressing cells exhibit an elevated level of hypophosphorylated form of pRb, and reduced level of cyclin B1 and p55CDC. ^ Our data suggest that p202 is a growth inhibitor gene in prostate cancer cells and its expression may also suppress transformation phenotype and tumorigenicity of prostate cancer cells. ^ In addition to inhibiting in vitro cell growth, suppressing the tumorigenicity of breast cancer cells in vivo, p202 expression could sensitize breast cancer cells to apoptosis induced by TNF-α treatment. One possible mechanism contributing to this sensitization is the inactivation of NF-κB by its interaction with p202. These results provide a scientific basis for a novel therapeutic strategy that combines p202 and TNF-α treatment against breast cancer. ^ It has been reported that NF-κB is constitutively active in human pancreatic cancer cells. Since p202 interacts with NF-κB and inhibits its activity, we examined a potential p202-mediated anti-tumor activity in pancreatic cancer. We used both ectopic and orthotopic xenograft models and demonstrated that p202 expression is associated with multiple anti-tumor activities that include inhibition of tumor growth, reduced tumorigenicity, prolonged survival, and remarkably, suppression of metastasis and angiogenesis. In vitro invasion assay also showed that p202-expressing pancreatic cancer cells are less invasive than those without p202 expression. That observation was supported by the findings that p202-expressing tumors showed reduced expression of angiogenic factors such as IL-8, and VEGF by inhibiting their transcription, and p202-expressing pancreatic cancer cells have reduced level of MAP-2 activity, a secreted protease activity important for metastasis. Together, our results strongly suggest that p202 expression mediates multiple anti-tumor activities against pancreatic cancer, and that may provide a scientific basis for developing a p202-based gene therapy in pancreatic cancer treatment. ^ Importantly, we demonstrated a treatment efficacy by using p202/SN2 liposome complex in a nude mice orthotopic breast cancer, and an ectopic pancreatic cancer xenograft model, through systemic and intra-tumor injection respectively. These results suggest a feasibility of using p202/SN2 liposome in future pre-clinical gene therapy experiments. ^

Dimethylsphingosine evokes diverse cellular death modes in HL60

Among the barriers to successful cancer treatment is the acquired resistance that tumors undergo due to clonal evolution. Non-cross resistant drugs could add to the current options of chemotherapeutic drugs. In order to improve tumor response, investigators have been identifying defective death pathways acquired by specific cancer types, so to target directly those pathways. Sphingolipids have emerged as potential drugs for tumor-targeted therapy, and among them, Dimethylsphingosine (DMS), known to be a competitive inhibitor of Sphingosine Kinase (SK). DMS actions have been documented by several investigators, but the mechanisms by which DMS exerts cytotoxicity have not been fully investigated. We evaluated the cytotoxicity of DMS against human leukemia cell lines and against blasts isolated from leukemia patients. Cell line viability decreased proportionally to DMS concentration and treatment time. Resistant and MDR positive cell lines were the most sensitive, indicating DMS efficacy against human leukemia MDR. Importantly, leukemia samples showed a similar sensitivity to DMS, the first demonstration of DMS activity against fresh human leukemia specimens. Mechanistically we have demonstrated that DMS efficacy is due to its ability to induce cytotoxicity by inducing necrosis, apoptosis or both concomitantly, revealing a mixed-feature cell death mode never described before for DMS. Further, we have shown evidence suggesting pathways cross-talk, since apoptosis inhibition led to accelerated rate of necrosis. DMS diverse killing mechanisms and the high expression of SK in leukemias could explain DMS potent cytotoxicity. DMS-based regimens may increase response rates and therefore, improve leukemia treatment. ^

A new fork for clinical application: Targeting forkhead transcription factors in cancer therapy

RAS-ERK-MAPK (Mitogen-activated protein kinase) pathway plays an essential role in proliferation, differentiation, and tumor progression. In this study, we showed that ERK downregulated FOXO3a through directly interacting with and phosphorylating FOXO3a at Serine 294, Serine 344, and Serine 425. ERK-phosphorylated FOXO3a was degraded by MDM2-mediated ubiquitin-proteosome pathway. FOXO3a phosphorylation and degradation consequently promoted cell proliferation and tumorigenesis. However, the non-phosphorylated FOXO3a mutant, which was resistant to the interaction and degradation by MDM2, resulted in inhibition of tumor formation. Forkhead O transcription factors (FOXOs) are important in the regulation of cellular functions including cell cycle arrest and cell death. Perturbation of FOXOs function leads to deregulated cell proliferation and cancer. Inactivation of FOXO proteins by activation of cell survival pathways, such as PI3K/AKT/IKK, is associated with tumorigenesis. Our study will further highlight FOXOs as new therapeutic targets in a broad spectrum of cancers. ^ Chemotherapeutic drug resistance is the most concerned problem in cancer therapy as resistance ultimately leads to treatment failure of cancer patients. In another study, we showed that blocking ERK activity with AZD6244, an established MEK1/2 inhibitor currently under human cancer clinical trials, enhances FOXO3a expression in various human cancer cell lines in vitro, and also in human colon cancer cell xenografts in vivo. Knocking down FOXO3a and its downstream gene Bim impaired AZD6244-induced growth suppression, whereas restoring activation of FOXO3a sensitized human cancer cell to AZD6244-induced growth arrest and apoptosis. More importantly, AZD6244-resistant cancer cells showed impaired endogenous FOXO3a nuclear translocation, reduced FOXO3a-Bim promoter association and significantly decreased Bim expression in response to AZD6244. AZD6244-resistant cancer cells can be sensitized to API-2 (an AKT inhibitor) and LY294002 (a PI3K inhibitor) in suppressing cell growth and colony formation, these inhibitors were known to enhance FOXO3a activity/nuclear translocation through inhibiting PI3K-AKT pathway. This study reveals novel molecular mechanism contributing to AZD6244-resistance through regulation of FOXO3a activity, further provides significant clinical implication of combining AZD6244 with PI3K/AKT inhibitors for sensitizing AZD6244-resistant cancer cells by activating FOXO3a. FOXO3a activation can be an essential pharmacological target and indicator to mediate and predict AZD6244 efficacy in clinical use. ^

Induction of synthetic lethality in mutant KRAS cells for non-small cell lung cancers chemoprevention and therapy

Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Despite improvement in treatment strategies, the 5-year survival rate of lung cancer patients remains low. Thus, effective chemoprevention and treatment approaches are sorely needed. Mutations and activation of KRAS occur frequently in tobacco users and the early stage of development of non-small cell lung cancers (NSCLC). So they are thought to be the primary driver for lung carcinogenesis. My work showed that KRAS mutations and activations modulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors by up-regulating death receptors and down-regulating decoy receptors. In addition, we showed that KRAS suppresses cellular FADD-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) expression through activation of ERK/MAPK-mediated activation of c-MYC which means the mutant KRAS cells could be specifically targeted via TRAIL induced apoptosis. The expression level of Inhibitors of Apoptosis Proteins (IAPs) in mutant KRAS cells is usually high which could be overcome by the second mitochondria-derived activator of caspases (Smac) mimetic. So the combination of TRAIL and Smac mimetic induced the synthetic lethal reaction specifically in the mutant-KRAS cells but not in normal lung cells and wild-type KRAS lung cancer cells. Therefore, a synthetic lethal interaction among TRAIL, Smac mimetic and KRAS mutations could be used as an approach for chemoprevention and treatment of NSCLC with KRAS mutations. Further data in animal experiments showed that short-term, intermittent treatment with TRAIL and Smac mimetic induced apoptosis in mutant KRAS cells and reduced tumor burden in a KRAS-induced pre-malignancy model and mutant KRAS NSCLC xenograft models. These results show the great potential benefit of a selective therapeutic approach for the chemoprevention and treatment of NSCLC with KRAS mutations.

Circumvention of cellular defense responses to DNA -directed nucleoside analogues by the protein kinase inhibitor, UCN-01

DNA-directed nucleoside analogues, such as ara-C, fludarabine, and gemcitabine, are antimetabolites effective in the treatment of a variety of cancers. However, resistance to nucleoside analogue-based chemotherapy in treatments is still a major problem in therapy. Therefore, it is essential to develop rationales for optimizing the use of nucleoside analogues in combination with other anticancer drugs or modalities such as radiation. The present study focuses on establishing mechanism-based combination strategy to overcome resistance to nucleoside analogues. ^ I hypothesized that the cytostatic concentrations of nucleoside analogues may cause S-phase arrest by activating an S-phase checkpoint that consists of a series of kinases. This may allow cells to repair damaged DNA over time and spare cytotoxicity. Thus, the ability of cells to enact an S-phase arrest in response to incorporation of potentially lethal amounts of nucleoside analogue may serve as a mechanism of resistance to S-phase-specific agents. As a corollary, the addition of a kinase inhibitor, such as UCN-01, may dysregulate the checkpoint response and abrogate the survival of S-phase-arrested cells by suppression of the survival signaling pathways. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, I demonstrated that cells arrested in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium, suggesting S-phase arrest served as a resistance mechanism of cancer cells to spare cytotoxicity of nucleoside analogues. However, nontoxic concentrations of UCN-01 rapidly killed S-phase-arrested cells by apoptosis. Furthermore, the molecular mechanism for UCN-01-induced apoptosis in S-phase-arrested cells was through inhibition of survival pathways associated with these cells. In this regard, suppression of the PI 3-kinase-Akt-Bad survival pathway as well as the NF-κB signaling pathway were associated with induction of apoptosis in S-phase-arrested cells by UCN-01, whereas the Ras-Raf-MEK-ERK pathway appeared not involved. This study has provided the rationales and strategies for optimizing the design of effective combination therapies to overcome resistance to nucleoside analogues. In fact, a clinical trial of the combination of ara-C with UCN-01 to treat relapsed or refractory AML patients has been initiated at U.T.M.D. Anderson Cancer Center. ^