Cell Survival Mechanisms Research Articles

Biphasic activation of extracellular signal-regulated kinase (ERK) 1/2 in epidermal growth factor (EGF)-stimulated SW480 colorectal cancer cells

Cancer cells have different characteristics due to the genetic differences where these unique features may strongly influence the effectiveness of therapeutic interventions. Here, we show that the spontaneous reactivation of extracellular signalregulated kinase (ERK), distinct from conventional ERK activation, represents a potent mechanism for cancer cell survival. We studied ERK1/2 activation in vitro in SW480 colorectal cancer cells. Although ERK signaling tends to be transiently activated, we observed the delayed reactivation of ERK1/2 in epidermal growth factor (EGF)-stimulated SW480 cells. This effect was observed even after EGF withdrawal. While phosphorylated ERK1/2 translocated into the nucleus following its primary activation, it remained in the cytoplasm during late-phase activation. The inhibition of primary ERK1/2 activation or protein trafficking, blocked reactivation and concurrently increased caspase 3 activity. Our results suggest that the biphasic activation of ERK1/2 plays a role in cancer cell survival; thus, regulation of ERK1/2 activation may improve the efficacy of cancer therapies that target ERK signaling. [BMB Reports 2016; 49(4): 220-225]

BRCA1 Gene Mutations and Influence of Chemotherapy on Autophagy and Apoptotic Mechanisms in Egyptian Breast Cancer Patients.

It is well established that mutations in the BRCA1 gene are a major risk factor for breast cancer. Induction of cancer cell death and inhibition of survival are the main principles of cancer therapy. In this context, autophagy may have dual roles in cancer, acting on the one hand as a tumor suppressor and on the other as a mechanism of cell survival that can promote the growth of established tumors. Therefore, understanding the role of autophagy in cancer treatment is critical. Moreover, defects in apoptosis, programmed cell death, may lead to increased resistance to chemotherapy. The aim of the present study was to detect BRCA1 gene mutations in order to throw more light on their roles as risk factors for breast cancer in Egypt. Secondly the role of autophagy and apoptosis in determining response to a fluorouracil, doxorubicin, cyclophosphamide (FAC) regimen was investigated. Forty-five female breast cancer cases and thirty apparently healthy females were enrolled in the present study. Serum levels of autophagic biomarkers, Beclin 1 and LC3 as well as the serum levels of apoptosis biomarkers Bcl-2 and Caspase-3 were measured before and after chemotherapy. BRCA1 mutations were found in 5 (16.7%) and 44 (99.8%) of the controls and cancer patients, the most frequent being 5382insC followed by C61G and 185 delAG. The results revealed that chemotherapy caused elevation in serum concentration levels of the autophagic biomarkers (Beclin 1 and LC3). This elevation was associated with a significant decrease in serum concentration levels of Bcl-2 and significant increase in caspase-3 concentration levels (apoptotic markers). The results of the present study indicate a very high level of BRCA mutations in breast cancer cases in Egypt and point to involvement of autophagic and apoptotic machinery activation in response to FAC chemotherapy.

Targeting CK2-driven non-oncogene addiction in B-cell tumors.

Genetic mutations of oncogenes often underlie deranged cell growth and altered differentiation pathways leading to malignant transformation of B-lymphocytes. However, addiction to oncogenes is not the only drive to lymphoid tumor pathogenesis. Dependence on non-oncogenes, which act by propelling basic mechanisms of cell proliferation and survival, has also been recognized in the pathobiology of lymphoid leukemias, lymphomas and multiple myeloma. Among the growing number of molecules that may uphold non-oncogene addiction, a key place is increasingly being recognized to the serine-threonine kinase CK2. This enzyme is overexpressed and overactive in B-acute lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia and non-Hodgkin lymphomas, such as mantle cell, follicular, Burkitt's and diffuse large B-cell lymphomas. In these tumors, CK2 may serve the activity of oncogenes, similar to BCR-ABL and c-MYC, control the activation of critical signaling cascades, such as NF-κB (nuclear factor-κB), STAT3 (signal transducer and activator of transcription 3) and PTEN/PI3K/AKT (phosphatase and tensin homolog protein/phosphoinositide 3-kinase/AKR thymoma), and sustain multiple cellular stress-elicited pathways, such as the proteotoxic stress, unfolded protein and DNA-damage responses. CK2 has also been shown to have an essential role in tuning signals derived from the stromal tumor microenvironment. Not surprisingly, targeting CK2 in lymphoid tumor cell lines or mouse xenograft models can boost the cytotoxic effects of both conventional chemotherapeutics and novel agents, similar to heat-shock protein 90, proteasome and tyrosine kinases inhibitors. In this review, we summarize the evidence indicating how CK2 embodies most of the features of a cancer growth-promoting non-oncogene, focusing on lymphoid tumors. We further discuss the preclinical data of the use of small ATP-competitive CK2 inhibitors, which hold the promise to be additional options in novel drug combinations for the therapy of lymphoid and plasmacellular malignancies.

Sa1801 Autonomous Nucleolar Mechanism of Colon Cancer Cell Survival and the Role of the Perinucleus in Ribosomal Protein Translation

Sa1801 Autonomous Nucleolar Mechanism of Colon Cancer Cell Survival and the Role of the Perinucleus in Ribosomal Protein Translation

Computational Analysis of the FLFP Motif in the Cell Survival Mechanism Conferred by PKCδVIII

Protein Kinase C δ (PKCδ) is a member of the novel group of serine/threonine kinases. In humans, PKCδ exists as two isoforms with opposite effects; PKCδI is pro‐apoptotic while PKCδVIIII is a cell survival regulator. PKCδVIII varies from the canonical sequence by a 31 amino acid insertion into the hinge region of PKCδI. A highly conserved hydrophobic linear motif, FLFP, was identified in this 31 amino acid variant region that we hypothesize contributes to the pro‐survival properties of PKCδVIII. Our lab has previously demonstrated that PKCδVIII forms complexes with mitogen activated protein kinase 1 (MAPK1) and Cyclin D1. However the exact mechanism of interaction has yet to be elucidated. Work in other model systems has demonstrated MAPK1 and Cyclin D1 to interact with the FLFP motif of other proteins. Thus, it is proposed that the interactions with PKCδVIII occur at the FLFP motif. Computational analyses of the impact site‐directed mutagenesis of the FLFP motif was performed to further investigate these protein complexes. Theoretical structures of PKCδVIII (wild‐type, F339Y, L340T, F341Y, and P342N) were created using I‐TASSER; Theoretical structures of PKCδVIII utilized in docking simulations were created using QUARK. YASARA was utilized to simulate the molecular docking of PKCδVIII to MAPK1 and Cyclin D1. The binding energies resulting from the theoretical docking were analyzed and compared to the wild‐type. Subsequent site‐directed mutagenesis of the FLFP motif has contributed to further clarifying our understanding of this protein complex. The results lend further evidence to support the role of PKCδVIII in promoting cellular survival.

Bacterial Control of Pores Induced by the Type III Secretion System: Mind the Gap.

Type III secretion systems (T3SSs) are specialized secretion apparatus involved in the virulence of many Gram-negative pathogens, enabling the injection of bacterial type III effectors into host cells. The T3SS-dependent injection of effectors requires the insertion into host cell membranes of a pore-forming “translocon,” whose effects on cell responses remain ill-defined. As opposed to pore-forming toxins that damage host cell plasma membranes and induce cell survival mechanisms, T3SS-dependent pore formation is transient, being regulated by cell membrane repair mechanisms or bacterial effectors. Here, we review host cell responses to pore formation induced by T3SSs associated with the loss of plasma membrane integrity and regulation of innate immunity. We will particularly focus on recent advances in mechanisms controlling pore formation and the activity of the T3SS linked to type III effectors or bacterial proteases. The implications of the regulation of the T3SS translocon activity during the infectious process will be discussed.

Autophagy: a new target for nonalcoholic fatty liver disease therapy.

Nonalcoholic fatty liver disease (NAFLD) has gained importance in recent decades due to drastic changes in diet, especially in Western countries. NAFLD occurs as a spectrum from simple hepatic steatosis, steatohepatitis to cirrhosis, and even hepatocellular carcinoma. Although the molecular mechanisms underlying the development of NAFLD have been intensively investigated, many issues remain to be resolved. Autophagy is a cell survival mechanism for disposing of excess or defective organelles, and has become a hot spot for research. Recent studies have revealed that autophagy is linked to the development of NAFLD and regulation of autophagy has therapeutic potential. Autophagy reduces intracellular lipid droplets by enclosing them and fusing with lysosomes for degradation. Furthermore, autophagy is involved in attenuating inflammation and liver injury. However, autophagy is regarded as a double-edged sword, as it may also affect adipogenesis and adipocyte differentiation. Moreover, it is unclear as to whether autophagy protects the body from injury or causes diseases and even death, and the association between autophagy and NAFLD remains controversial. This review is intended to discuss, comment, and outline the progress made in this field and establish the possible molecular mechanism involved.

Photodynamic Therapy Synergizes with Irinotecan to Overcome Compensatory Mechanisms and Improve Treatment Outcomes in Pancreatic Cancer.

The ability of tumor cells to adapt to therapeutic regimens by activating alternative survival and growth pathways remains a major challenge in cancer therapy. Therefore, the most effective treatments will involve interactive strategies that target multiple nonoverlapping pathways while eliciting synergistic outcomes and minimizing systemic toxicities. Nanoliposomal irinotecan is approved by the FDA for gemcitabine-refractory metastatic pancreatic cancer. However, the full potential of irinotecan treatment is hindered by several cancer cell survival mechanisms, including ATP-binding cassette G2 (ABCG2) transporter-mediated irinotecan efflux from cells. Here, we demonstrate that benzoporphyrin derivative-based photodynamic therapy (PDT), a photochemical cytotoxic modality that activates the apoptotic pathway, reduced ABCG2 expression to increase intracellular irinotecan levels in pancreatic cancer. Moreover, we show that PDT inhibited survivin expression. Although PDT potentiated irinotecan treatment, we also demonstrate that irinotecan reduced the tumoral expression of monocarboxylate transporter 4, which was upregulated by PDT. Notably, using orthotopic xenograft models, we demonstrate that combination of single low-dose PDT and a subclinical dose of nanoliposomal irinotecan synergistically inhibited tumor growth by 70% for 3 weeks compared with 25% reduction after either monotherapies. Our findings offer new opportunities for the clinical translation of PDT and irinotecan combination therapy for effective pancreatic cancer treatment.

Coordinated induction of cell survival signaling in the inflamed microenvironment of the prostate.

Both prostate cancer and benign prostatic hyperplasia are associated with inflammatory microenvironments. Inflammation is damaging to tissues, but it is unclear how the inflammatory microenvironment protects specialized epithelial cells that function to proliferate and repair the tissue. The objective of this study is to characterize the cell death and cell survival response of the prostatic epithelium in response to inflammation. We assessed induction of cell death (TNF, TRAIL, TWEAK, FasL) and cell survival factors (IGFs, hedgehogs, IL-6, FGFs, and TGFs) in inflamed and control mouse prostates by ELISA. Cell death mechanisms were determined by immunoblotting and immunofluorescence for cleavage of caspases and TUNEL. Survival pathway activation was assessed by immunoblotting and immunofluorescence for Mcl-1, Bcl-2, Bcl-XL, and survivin. Autophagy was determined by immunoblotting and immunofluorescence for free and membrane associated light chain 3 (LC-3). Cleavage of all four caspases was significantly increased during the first 2 days of inflammation, and survival protein expression was substantially increased subsequently, maximizing at 3 days. By 5 days of inflammation, 50% of prostatic epithelial cells expressed survivin. Autophagy was also evident during the recovery phase (3 days). Finally, immunofluorescent staining of human specimens indicates strong activation of survival proteins juxtaposed to inflammation in inflamed prostate specimens. The prostate responds to deleterious inflammation with induction of cell survival mechanisms, most notably survivin and autophagy, demonstrating a coordinated induction of survival factors that protects and expands a specialized set of prostatic epithelial cells as part of the repair and recovery process during inflammation.

Adoptive Autophagy Activation: a Much-Needed Remedy Against Chemical Induced Neurotoxicity/Developmental Neurotoxicity.

The profound significance of autophagy as a cell survival mechanism under conditions of metabolic stress is a well-proven fact. Nearly a decade-long research in this area has led scientists to unearth various roles played by autophagy other than just being an auto cell death mechanism. It is implicated as a vital cell survival pathway for clearance of all the aberrant cellular materials in case of cellular injury, metastasis, disease states, cellular stress, neurodegeneration and so on. In this review, we emphasise the critical role of autophagy in the environmental stressors-induced neurotoxicity and its therapeutic implications for the same. We also attempt to shed some light on the possible protective role of autophagy in developmental neurotoxicity (DNT) which is a rapidly growing health issue of the human population at large and hence a point of rising concern amongst researchers. The intimate association between DNT and neurodegenerative disorders strongly indicates towards adopting autophagy activation as a much-needed remedy for DNT.

Routine molecular testing of resected early-stage lung adenocarcinoma with targeted next-generation sequencing demonstrates a high rate of actionable mutations

Cell surface mono-ADP ribosyltransferases (ARTs) transfer the ADP-ribose moiety from NADþ to amino acid residues on target proteins and post-translationally regulate their function. Most mammalian mono-ARTs, including ADP-ribosyltransferase 1 (ART1) reside on the cell surface, however the scope of extracellular monoADP-ribosylation is largely unknown. It has been suggested that epithelial cells in the injured or inflamed lung may overexpress ART1 as a mechanism of cell survival to protect against cell clearance by inflammatory cells. We hypothesize that ART1 expression is cytoprotective to lung cancer cells and facilitates metastatic growth. We found ART1 to be expressed in multiple human NSCLC cell lines of distinct driver mutation status by RTPCR, western blots, and immunofluorescent staining. Using biobanked human materials, we also found evidence of ART1 expression in human NSCLC tumors by whole tumor RT-PCR, immunofluorescence, and immunohistochemistry. Compared tomatchedadjacent normal lung (n1⁄440), byRTPCR there is over a 2-fold increase (p1⁄40.01) in median tumor expression of ART1, suggesting a role in tumorigenesis or tumor progression. We subsequently stained a tissue microarray containing 184 cases of predominantly (74%) stage I NSCLC to determine the prevalence of NSCLC tumors staining positive for ART1. ART1 staining was moderate or strong in 83% of adenocarcinomas (n1⁄4145) and in 45% of squamous cell cancers (n1⁄439, p<0.001). We next used murine cell lines derived from inducible KRASG12D/þ/p53-/mice and an in vivo model to determine the effect of ART1 expression onmetastatic outgrowth. In a tail vein injection model in immunocompetent mice, we noted a highly significant decrease in metastasis in the ART1-knockdown cell line (sh175) compared to the parent KP1 line. To determine whether the immune protective effect mediated by ART1 may be T cell dependent, we implanted flank tumors in immunocompetent (B6) and lymphocyte-depleted nude mice. Despite slightly faster in vitro rates of growth, sh175KP1 cells lacking ART1 expression were largely unable to form tumors when injected into the flanks of immunocompetent B6 mice. Only 1 of 5 immunocompetent mice injected with sh175KP1 cells developed a tumor, compared to 5 of 5 mice injected with KP1 control cells. However, in lymphocyte-depleted nude mice, there was robust flank tumor growth with both cell lines. Because the sh175KP1 cells lacking ART1 only had a disadvantage to growth in mice with lymphocytes, we can infer that ART1 expression may have a strong immunomodulating effect on that cell population.We also evaluated the effect of ART1 expression on neutrophil cytotoxicity. At a neutrophil:tumor cell ratio of 20:1, the knockdown cell line sh175KP1 lacking ART1 expression is more sensitive to neutrophil-induced apoptosis in the co-culture assay (87% vs. 56% Annexin V positive, p1⁄40.05). Importantly, chemical inhibition of mono-ADP-ribosylation in the parent KP1 cell line with two well established inhibitors, novobiocin and meta-iodobenzylguanidine, facilitated neutrophil-induced apoptosis, implying that the enzymatic activity of ART1 is critical to the phenotype. We provide evidence that ART1 is overexpressed in NSCLC. ADP-ribosylation may serve as a defense against immune-mediated cytotoxicity. Cells without ART1 expression are more sensitive to cytotoxicity induced by immune cells and these cells have a markedly decreased capacity to grow in the lungs in an immunocompetentmetastaticmodel. Because ART1 is an extracellular enzymatic target, it is expected to be highly druggable and susceptible to therapeutic intervention. We envision using targeted inhibition of ADPribosylation in ART1-overexpressing tumors to facilitate immune-mediated destruction of established cancers or of micrometastatic disease.

ART1, an extracellular ADP-ribosyltransferase, is over-expressed in non-small cell lung cancer and facilitates cancer cell survival by immune-mediated mechanisms

Cell surface mono-ADP ribosyltransferases (ARTs) transfer the ADP-ribose moiety from NADþ to amino acid residues on target proteins and post-translationally regulate their function. Most mammalian mono-ARTs, including ADP-ribosyltransferase 1 (ART1) reside on the cell surface, however the scope of extracellular monoADP-ribosylation is largely unknown. It has been suggested that epithelial cells in the injured or inflamed lung may overexpress ART1 as a mechanism of cell survival to protect against cell clearance by inflammatory cells. We hypothesize that ART1 expression is cytoprotective to lung cancer cells and facilitates metastatic growth. We found ART1 to be expressed in multiple human NSCLC cell lines of distinct driver mutation status by RTPCR, western blots, and immunofluorescent staining. Using biobanked human materials, we also found evidence of ART1 expression in human NSCLC tumors by whole tumor RT-PCR, immunofluorescence, and immunohistochemistry. Compared tomatchedadjacent normal lung (n1⁄440), byRTPCR there is over a 2-fold increase (p1⁄40.01) in median tumor expression of ART1, suggesting a role in tumorigenesis or tumor progression. We subsequently stained a tissue microarray containing 184 cases of predominantly (74%) stage I NSCLC to determine the prevalence of NSCLC tumors staining positive for ART1. ART1 staining was moderate or strong in 83% of adenocarcinomas (n1⁄4145) and in 45% of squamous cell cancers (n1⁄439, p<0.001). We next used murine cell lines derived from inducible KRASG12D/þ/p53-/mice and an in vivo model to determine the effect of ART1 expression onmetastatic outgrowth. In a tail vein injection model in immunocompetent mice, we noted a highly significant decrease in metastasis in the ART1-knockdown cell line (sh175) compared to the parent KP1 line. To determine whether the immune protective effect mediated by ART1 may be T cell dependent, we implanted flank tumors in immunocompetent (B6) and lymphocyte-depleted nude mice. Despite slightly faster in vitro rates of growth, sh175KP1 cells lacking ART1 expression were largely unable to form tumors when injected into the flanks of immunocompetent B6 mice. Only 1 of 5 immunocompetent mice injected with sh175KP1 cells developed a tumor, compared to 5 of 5 mice injected with KP1 control cells. However, in lymphocyte-depleted nude mice, there was robust flank tumor growth with both cell lines. Because the sh175KP1 cells lacking ART1 only had a disadvantage to growth in mice with lymphocytes, we can infer that ART1 expression may have a strong immunomodulating effect on that cell population.We also evaluated the effect of ART1 expression on neutrophil cytotoxicity. At a neutrophil:tumor cell ratio of 20:1, the knockdown cell line sh175KP1 lacking ART1 expression is more sensitive to neutrophil-induced apoptosis in the co-culture assay (87% vs. 56% Annexin V positive, p1⁄40.05). Importantly, chemical inhibition of mono-ADP-ribosylation in the parent KP1 cell line with two well established inhibitors, novobiocin and meta-iodobenzylguanidine, facilitated neutrophil-induced apoptosis, implying that the enzymatic activity of ART1 is critical to the phenotype. We provide evidence that ART1 is overexpressed in NSCLC. ADP-ribosylation may serve as a defense against immune-mediated cytotoxicity. Cells without ART1 expression are more sensitive to cytotoxicity induced by immune cells and these cells have a markedly decreased capacity to grow in the lungs in an immunocompetentmetastaticmodel. Because ART1 is an extracellular enzymatic target, it is expected to be highly druggable and susceptible to therapeutic intervention. We envision using targeted inhibition of ADPribosylation in ART1-overexpressing tumors to facilitate immune-mediated destruction of established cancers or of micrometastatic disease.

Abstract PR18: Survival of ARHI-induced dormant autophagic cell death in vivo requires permissive levels of VEGF, IL-8, and IGF-1 in the tumor microenvironment.

Abstract Among gynecological cancers, ovarian cancer has the worst prognosis. One of the most important factors contributing to poor outcomes is the ability of drug resistant ovarian cancer cells to remain dormant for years after treatment, only to grow progressively and kill their host. Judging from sites of recurrence, most dormant ovarian cancer cells are found on the surface of the peritoneal cavity, often in small, poorly vascularized collagenous scars observed at second look operations. Our group has found that autophagy and tumor dormancy can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), which is downregulated in 60% of ovarian cancers and in cancers from several other sites. Re-expression of ARHI induces autophagy by inhibiting PI3K and mTOR signaling, displacing Bcl-2 from Beclin1to form the autophagy initiating complex, inducing ATG4, decorating the autophagosome membrane and co-localizing with LC3II. Re-expression of ARHI in ovarian cancer cells in culture produces cell death within 72 hours, whereas re-expression of ARHI in xenografts produces cell growth arrest and tumor dormancy. When ARHI levels are reduced once again in vivo, xenografts grow promptly to kill the host. Outgrowth of dormant xenografts is markedly delayed, but not prevented by treatment with chloroquine, a functional inhibitor of autophagy. The clinical relevance of this dormancy model is supported by previous studies that found ARHI expression in ovarian cancer cells was significantly increased in 90% of cases where small, dormant nodules of drug resistant ovarian cancer removed from the peritoneal surface at second-look operations, but in only 40% of the primary specimens from the same patients from initial cytoreductive surgery. In this study, we have measured Ki67 and microvascular density (MVD) in the same set of primary human ovarian cancers and in matched positive second look specimens. Second look specimens exhibited significantly lower Ki-67 (p&amp;lt;0.0001) and MVD (p&amp;lt;0.0001) than did primary cancers. We have hypothesized that that decreased access to nutrients with decreased MVD could be responsible the elevation of ARHI levels and the induction of autophagy. While autophagy could sustain nutrient deprived cancer cells up to a point, excessive autophagy can also induce type II autophagic cancer cell death. Our previous studies indicated that growth factors (VEGF, IGF1) and cytokines (IL-8) present in the tumor microenvironment could rescue ovarian cancer cells in culture from autophagic death. In this study we have found that treatment with anti-VEGF, anti-IL8 and anti-IGFR1 antibodies, individually and in combination, slowed the outgrowth of dormant autophagic ovarian cancer cells. Treatment of dormant xenografts with the same monoclonal antibodies against VEGF, IL-8 and IGFR1 with or without CQ not only delayed outgrowth when DOX is withdrawn, but cured a fraction of mice. Re-expression of ARHI reduced expression of IGF-1 receptor, but not VEGF and IL8 receptors. Treatment ovarian cancer cells with VEGF, IL-8 and IGF-1 reduced ARHI's ability inducing autophagy and inhibiting AKT and mTOR activity. Our studies provide a novel model for tumor dormancy that incorporates autophagy as a mechanism for tumor cell survival in the presence of permissive levels of growth factors and cytokines. Blocking these survival factors with antibodies could provide a novel approach to eliminate dormant cells, providing more effective treatment for women with ovarian cancer. This abstract is also presented as Poster A36. Citation Format: Zhen Lu, Aaron F. Orozco, Hailing Yang, Margie N. Sutton, Yan Wang, Weiqun Mao, Douglas A. Levine, Robert C. Bast, Jr., Robert C. Bast, Jr.. Survival of ARHI-induced dormant autophagic cell death in vivo requires permissive levels of VEGF, IL-8, and IGF-1 in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR18.

Clinical trial designs incorporating predictive biomarkers.

Development of oncologic therapies has traditionally been performed in a sequence of clinical trials intended to assess safety (phase I), preliminary efficacy (phase II), and improvement over the standard of care (phase III) in homogeneous (in terms of tumor type and disease stage) patient populations. As cancer has become increasingly understood on the molecular level, newer "targeted" drugs that inhibit specific cancer cell growth and survival mechanisms have increased the need for new clinical trial designs, wherein pertinent questions on the relationship between patient biomarkers and response to treatment can be answered. Herein, we review the clinical trial design literature from initial to more recently proposed designs for targeted agents or those treatments hypothesized to have enhanced effectiveness within patient subgroups (e.g., those with a certain biomarker value or who harbor a certain genetic tumor mutation). We also describe a number of real clinical trials where biomarker-based designs have been utilized, including a discussion of their respective advantages and challenges. As cancers become further categorized and/or reclassified according to individual patient and tumor features, we anticipate a continued need for novel trial designs to keep pace with the changing frontier of clinical cancer research.

Inhibition of HSP90 Promotes Neural Stem Cell Survival from Oxidative Stress through Attenuating NF-κB/p65 Activation.

Stem cell survival after transplantation determines the efficiency of stem cell treatment, which develops as a novel potential therapy for several central nervous system (CNS) diseases in recent decades. The engrafted stem cells face the damage of oxidative stress, inflammation, and immune response at the lesion point in host. Among the damaging pathologies, oxidative stress directs stem cells to apoptosis and even death through several signalling pathways and DNA damage. However, the in-detail mechanism of stem cell survival from oxidative stress has not been revealed clearly. Here, in this study, we used hydrogen peroxide (H2O2) to induce the oxidative damage on neural stem cells (NSCs). The damage was in consequence demonstrated involving the activation of heat shock protein 90 (HSP90) and NF-κB/p65 signalling pathways. Further application of the pharmacological inhibitors, respectively, targeting at each signalling indicated an upper-stream role of HSP90 upon NF-κB/p65 on NSCs survival. Preinhibition of HSP90 with the specific inhibitor displayed a significant protection on NSCs against oxidative stress. In conclusion, inhibition of HSP90 would attenuate NF-κB/p65 activation by oxidative induction and promote NSCs survival from oxidative damage. The HSP90/NF-κB mechanism provides a new evidence on rescuing NSCs from oxidative stress and also promotes the stem cell application on CNS pathologies.

ROS-Induced CXCR4 Signaling Regulates Mantle Cell Lymphoma (MCL) Cell Survival and Drug Resistance in the Bone Marrow Microenvironment via Autophagy.

Patients with advanced stages of mantle cell lymphoma (MCL) have a poor prognosis after standard therapies. MCL cells in those patients often spread into tissues other than lymph nodes, such as the bone marrow. Apart from directed migration and homing, there is little understanding of the function of the CXCR4/SDF-1 signaling axis in MCL. In this report, we aim to understand mechanisms of MCL cell survival in the bone marrow. For comprehensive analyses of MCL interactions with bone marrow stromal cells, we have generated gene knockout cells using CRISPR-CAS9 system and gene knockdown cells to reveal novel roles of the CXCR4/SDF-1 signaling. CXCR4 silencing in MCL cells led to a significant reduction in proliferation, cell adhesion to bone marrow stromal cells, and colony formation in PHA-LCM methylcellulose medium, which were reversed upon the addition of SDF-1-neutralizing antibodies. In addition, tracking MCL cell engraftment in vivo revealed that quiescent MCL cells are significantly reduced in the bone marrow upon CXCR4 silencing, indicating that CXCR4/SDF-1 signaling is required for the survival and maintenance of the quiescent MCL cells. Further analysis revealed novel mechanisms of ROS-induced CXCR4/SDF-1 signaling that stimulate autophagy formation in MCL cells for their survival. Our data, for the first time, revealed new roles of the CXCR/SDF-1 signaling axis on autophagy formation in MCL, which further promoted their survival within the bone marrow microenvironment. Targeting the CXCR4/SDF-1/autophagy signaling axis may contribute to an enhanced efficacy of current therapies.

200 Million Thymocytes and I: A Beginner's Survival Guide to T Cell Development.

T lymphocytes (T cells) are essential for proper adaptive immune responses. They perform a variety of functions in defenses against pathogens, and notably control, positively or negatively, other cells involved in immune responses. T cells develop in the thymus from bone marrow-derived precursors. These precursors (thymocytes) proliferate, rearrange the genes encoding subunits of the T cell antigen receptor, which endow them with their unique antigen specificity, and undergo various degrees of pre-programming for their functions in immune responses. Thus, analyzing T cell development in the thymus is essential for understanding their functions in immune responses. In addition, the thymus constitutes an attractive experimental model to analyze mechanisms of cell proliferation, differentiation and survival, all of which are involved in thymocyte development. This chapter presents a quick overview of the key events characterizing intrathymic T cell development, as an introduction for readers entering this field of study.

Abstract A91: Over-the-counter melatonin supplementation in human subjects: A potentially novel chronotherapeutic approach targeting the Warburg effect and fatty acid metabolism in breast cancer therapy/prevention

Abstract Melatonin, a circadian anti-cancer hormone produced by the pineal gland during darkness at night suppresses the Warburg effect, linoleic acid (LA) uptake/metabolism and tumor cell proliferation in both estrogen receptor (ERα+) and ERα- in tissue-isolated human breast cancer xenografts. The nighttime circadian melatonin signal regulates circadian rhythms in tumor glucose and fatty acid metabolism as well as related signaling pathways that are important in controlling cell proliferative and survival mechanisms. Over-the-counter (OTC) melatonin supplements are used by millions of individuals to treat insomnia and/or jet-lag. The present study addressed the hypothesis that oral ingestion of OTC melatonin supplements by normal adult human female volunteers results in blood levels of melatonin that suppress LA uptake/metabolism, aerobic glycolysis and cell proliferative activity in human breast cancer xenografts, growing in nude female rats, directly perfused in situ with human subject donor whole-blood following melatonin intake. Twelve young, healthy premenopausal women were recruited to ingest an OTC melatonin supplement at a single dose of either 75μg, 150μg, 300μg or 1 mg during midday (low endogenous melatonin levels) resulting in low to high pharmacological blood concentrations of melatonin. A pre-supplement venous blood sample was collected from the antecubital vein of the forearm. Each subject then ingested a randomly selected oral dose of melatonin followed approximately 1 hour later by the withdrawal of a post-supplement venous blood sample. On the following day, the pre- and post-supplement whole-blood samples collected from a given subject were separately placed into a tumor perfusion reservoir. Tissue-isolated ERα- MCF-7 human breast cancer xenografts grown in female nude rats were then directly perfused in situ for 1 hour with either pre- or post-supplement oxygenated blood (37°C). Irrespective of the dose tested, melatonin induced a 50% decrease in both tumor glucose uptake and lactate release, 22% and 44% deceases in O2 uptake and CO2 production, respectively, 50% decrease in cAMP concentrations, and a 100% decrease in linoleic acid (LA) uptake and 13-hydroxyoctadecadienoic acid (13-HODE) formation. Melatonin also caused a marked decrease in the expression of phospho-AKT, GSK3β and ERK1/2, and an 85% decrease in the incorporation of [3H]thymidine into DNA. Similar results were obtained at the lowest dose of melatonin (e.g., 75μg) in ERα+ human breast cancer xenografts. The melatonin-induced suppression tumor proliferative and metabolic activity in both ERα- and ERα+ breast cancer xenografts was completely prevented by the co-perfusion with the non-selective MT1/MT22 melatonin receptor blocker S20928 consistent with the involvement of a melatonin receptor-mediated mechanism. These results indicate that oral administration of an OTC melatonin supplement at a variety of low to high doses were equally effective in suppressing the Warburg effect and key tumor proliferative and survival signaling pathways, cAMP-dependent LA uptake and metabolism to mitogenically active 13-HODE and ultimately cell proliferation in tissue-isolated human breast xenografts irrespective of ERα status via a melatonin receptor-mediated mechanism. These findings suggest that even low doses of OTC melatonin preparations may play a potentially important role as a new chronotherapeutic agent in human breast cancer treatment and/or prevention by targeting aerobic glycolysis and fatty acid signaling and metabolism. Supported by NIH Grants R21CA129875 (DEB) and R01CA54152 (SMH). Citation Format: David E. Blask, Robert T. Dauchy, Erin M. Dauchy, Steven M. Hill, Lulu Mao, Melissa M. Wren, Mary M.C. Meyaski-Schluter, Lin Yuan. Over-the-counter melatonin supplementation in human subjects: A potentially novel chronotherapeutic approach targeting the Warburg effect and fatty acid metabolism in breast cancer therapy/prevention. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A91.

Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9.

Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit (1) based on a 4,6-pyrimidine scaffold (2.00 μM). Optimisation of cellular SAR led to an inhibitor with improved potency (25, 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM).

Effect of hypoxia on chemosensitivity to 5-fluorouracil in SH-SY5Y neuroblastoma cells

Resistance to chemotherapy is a major obstacle in clinical oncology. Hypoxia is a hallmark of solid tumours as a result of poorly structured tumour neovasculature and is known to be a key contributor to cancer malignancy and reduced drug effectiveness. Hypoxia-inducible factor-1 (HIF-1) mediates the hypoxic response, bringing about adaptive responses to the change in microenvironment. This study investigated the effect of 24 h hypoxia on the sensitivity of SH-SY5Y neuroblastoma cells to 5-fluorouracil (5-FU). Cytotoxicity, apoptosis and membrane integrity cell-based assays were carried out to observe changes in cell viability alongside apoptotic and necrotic cell death to determine the molecular mechanisms underlying reduced drug sensitivity. Hypoxic cells showed no change in cell viability to 5-FU in comparison to normoxic controls, suggesting that hypoxia conferred reduced drug sensitivity. As a measure of apoptosis, caspase-3/7 levels were significantly higher in hypoxic cells treated with increasing 5-FU concentrations. However, this was associated with less cell necrosis, suggesting that despite increased hypoxia-induced apoptosis, a significant decrease in uncontrolled cell death accounted for the change in viability observed. This finding also implies that alternative cell survival mechanisms could also be activated.