Tumor Cell Survival Mechanisms Research Articles

Cytoprotective, Cytotoxic and Cytostatic Roles of Autophagy in Response to BET Inhibitors.

The bromodomain and extra-terminal domain (BET) family inhibitors are small molecules that target the dysregulated epigenetic readers, BRD2, BRD3, BRD4 and BRDT, at various transcription-related sites, including super-enhancers. BET inhibitors are currently under investigation both in pre-clinical cell culture and tumor-bearing animal models, as well as in clinical trials. However, as is the case with other chemotherapeutic modalities, the development of resistance is likely to constrain the therapeutic benefits of this strategy. One tumor cell survival mechanism that has been studied for decades is autophagy. Although four different functions of autophagy have been identified in the literature (cytoprotective, cytotoxic, cytostatic and non-protective), primarily the cytoprotective and cytotoxic forms appear to function in different experimental models exposed to BET inhibitors (with some evidence for the cytostatic form). This review provides an overview of the cytoprotective, cytotoxic and cytostatic functions of autophagy in response to BET inhibitors in various tumor models. Our aim is to determine whether autophagy targeting or modulation could represent an effective therapeutic strategy to enhance the response to these modalities and also potentially overcome resistance to BET inhibition.

Peritoneal Spread of Ovarian Cancer Harbors Therapeutic Vulnerabilities Regulated by FOXM1 and EGFR/ERBB2 Signaling.

Peritoneal spread is the primary mechanism of metastasis of ovarian cancer, and survival of ovarian cancer cells in the peritoneal cavity as nonadherent spheroids and their adherence to the mesothelium of distant organs lead to cancer progression, metastasis, and mortality. However, the mechanisms that govern this metastatic process in ovarian cancer cells remain poorly understood. In this study, we cultured ovarian cancer cell lines in adherent and nonadherent conditions in vitro and analyzed changes in mRNA and protein levels to identify mechanisms of tumor cell survival and proliferation in adherent and nonadherent cells. EGFR or ERBB2 upregulated ZEB1 in nonadherent cells, which caused resistance to cell death and increased tumor-initiating capacity. Conversely, Forkhead box M1 (FOXM1) was required for the induction of integrin β1, integrin-α V, and integrin-α 5 for adhesion of cancer cells. FOXM1 also upregulated ZEB1, which could act as a feedback inhibitor of FOXM1, and caused the transition of adherent cells to nonadherent cells. Strikingly, the combinatorial treatment with lapatinib [dual kinase inhibitor of EGFR (ERBB1) and ERBB2] and thiostrepton (FOXM1 inhibitor) reduced growth and peritoneal spread of ovarian cancer cells more effectively than either single-agent treatment in vivo. In conclusion, these results demonstrate that FOXM1 and EGFR/ERBB2 pathways are key points of vulnerability for therapy to disrupt peritoneal spread and adhesion of ovarian cancer cells. SIGNIFICANCE: This study describes the mechanism exhibited by ovarian cancer cells required for adherent cell transition to nonadherent form during peritoneal spread and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5554/F1.large.jpg.

A Potent and Selective Dual Inhibitor of AXL and MERTK Possesses Both Immunomodulatory and Tumor-Targeted Activity.

TYRO3, AXL, and MERTK constitute the TAM family of receptor tyrosine kinases, which play important roles in tumor growth, survival, cell adhesion, as well as innate immunity, phagocytosis, and immune-suppressive activity. Therefore, targeting both AXL and MERTK kinases may directly impact tumor growth and relieve immunosuppression. We describe here the discovery of INCB081776, a potent and selective dual inhibitor of AXL and MERTK that is currently in phase 1 clinical trials. In cellular assays, INCB081776 effectively blocked autophosphorylation of AXL or MERTK with low nanomolar half maximal inhibitory concentration values in tumor cells and Ba/F3 cells transfected with constitutively active AXL or MERTK. INCB081776 inhibited activation of MERTK in primary human macrophages and partially reversed M2 macrophage–mediated suppression of T-cell proliferation, which was associated with increased interferon-γ production. In vivo, the antitumor activity of INCB081776 was enhanced in combination with checkpoint blockade in syngeneic models, and resulted in increased proliferation of intratumoral CD4+ and CD8+ T cells. Finally, antitumor activity of INCB081776 was observed in a subset of sarcoma patient–derived xenograft models, which was linked with inhibition of phospho-AKT. These data support the potential therapeutic utility of INCB081776 as an immunotherapeutic agent capable of both enhancing tumor immune surveillance and blocking tumor cell survival mechanisms.

Highlights of This Issue

Prevention or treatment of relapsed lymphoid malignancies after hematopoietic stem cell transplantation requires novel strategies. Khouri and colleagues show that the addition of lenalidomide to ipilimumab at 3 mg/kg for just two cycles can induce greater than expected and durable clinical responses. This impact also is suggested by the additive increased numbers of inducible costimulatory +CD4+ T cells by lenalidomide. The preliminary data indicate similar immune responses in the allogeneic and autologous transplant recipients. The treatment is safe, with no severe treatment-related toxicities, minimal immune-mediated toxicity and no graft-versus-host disease. Findings from this study support further investigation of this combination in this setting.Barrett's esophagus can be diagnosed and biopsied by esophagogastroduodenoscopy, but current screening and surveillance strategies are limited by the absence of specific imaging markers predictive of increased risk for esophageal adenocarcinomas (EAC). Fang and colleagues use a Barrett's esophagus mouse model and EAC patient data to demonstrate that the chemokine receptor 4 (CXCR4) is upregulated in the immune tumor microenvironment, thus presenting a highly attractive target for PET/CT imaging using CXCR4-targeted probes. These findings provide new insights into the kinetics and pathogenesis of EAC and introduce CXCR4-based imaging of the tumor microenvironment as a potential approach for diagnosis and surveillance of EAC.RAS-mutant cancer is difficult to treat with standard care. Nonsteroidal anti-inflammatory drugs like aspirin have been proposed to reduce cancer burden. Hammerlindl and colleagues describe combining aspirin with sorafenib, a multi-kinase inhibitor, in RAS-mutant melanoma and lung cancer. The combination induced activation of both AMPK and ERK pathways, leading to strongly enhanced tumor cytotoxicity at relatively low sorafenib doses. Combining aspirin and sorafenib is a viable treatment strategy in refractory and relapsed RAS-mutant cancer.Effective therapies for glioblastoma, a cancer known to being highly resistant to chemotherapy, may have to target multiple tumor cell survival mechanisms. Su and colleagues investigated the mechanisms of action of TG02, a novel CDK9 inhibitor, as a single agent and in combination with temozolomide, utilizing in vitro and in vivo glioblastoma models. In addition to transcriptional suppression, TG02 also was found to induce mitochondrial dysfunction, glycolytic suppression, and, when combined with temozolomide, synergistically decreased energy production in glioblastoma. These results have led to an ongoing clinical trial investigating this promising therapeutic strategy in patients with recurrent glioblastomas (NCT02942264).

Natural Killer Cells: Angels and Devils for Immunotherapy

In recent years, the relevance of the immune system to fight cancer has led to the development of immunotherapy, including the adoptive cell transfer of immune cells, such as natural killer (NK) cells and chimeric antigen receptors (CAR)-modified T cells. The discovery of donor NK cells’ anti-tumor activity in acute myeloid leukemia patients receiving allogeneic stem cell transplantation (allo-SCT) was the trigger to conduct many clinical trials infusing NK cells. Surprisingly, many of these studies did not obtain optimal results, suggesting that many different NK cell parameters combined with the best clinical protocol need to be optimized. Various parameters including the high array of activating receptors that NK cells have, the source of NK cells selected to treat patients, different cytotoxic mechanisms that NK cells activate depending on the target cell and tumor cell survival mechanisms need to be considered before choosing the best immunotherapeutic strategy using NK cells. In this review, we will discuss these parameters to help improve current strategies using NK cells in cancer therapy. Moreover, the chimeric antigen receptor (CAR) modification, which has revolutionized the concept of immunotherapy, will be discussed in the context of NK cells. Lastly, the dark side of NK cells and their involvement in inflammation will also be discussed.

Cell-Cell Communication Between Multiple Myeloma (MM) Cells and Cord Blood Derived NK Cells (CB-NK) Regulates Both Tumor Cell Death and Tumor Cell Survival

In previous work we have demonstrated that CB-NK exert a cytotoxicity towards MM cells which involves lipid-protein vesicle transfer, which it is secondarily transferred from recipient MM cells to neighboring MM cells. This transmissible cytotoxicity causes a 5-7% of neighboring MM cell death (CDD 2015;22(1):96-107). However, this transmission between MM cells could have a diluent effect of the CB-NK cytotoxicity which could be also a tumor cell survival mechanism. To further analyze this mechanism, we determined the proteins transferred between cells and the role of lipids in this transfer.We performed TRANS-SILAC proteomics using two different approaches: 1) Labeling CB-NK cells with heavy amino-acids (hAA) to identify both CB-NK proteins transferred to MM (1ºMM) and secondary CB-NK proteins transferred from 1ºMM to neighboring MM cells (2ºMM). 2) Labeling MM cells, before CB-NK contact, with hAA to identify transferred proteins from 1ºMM to 2ºMM cells, and from 1ºMM to CB-NK.We found that 1ºMM cells acquired 9.5% of CB-NK proteins, and that these proteins were transferred to 2ºMM cells. As a consequence, 1ºMM cells diluted the CB-NK proteins from 9.5% to 3.8%; which represented 7.2% of proteins in 2ºMM cells. In the second approach, we observed that, MM cells transferred to neighboring MM cells only 1.9% of MM proteins in resting conditions. However, in the presence of CB-NK, this transfer was increased up to 7.7%. Furthermore, CB-NK cells acquired 7.3% of 1ºMM proteins. These findings demonstrate a secondary CB-NK protein transfer between MM cells, which represents a CB-NK protein dilution content from 1ºMM to 2ºMM, and an increased transfer of 1ºMM proteins to 2ºMM (Fig.1A). Proteomic analysis showed that transferred proteins were involved in FAS signaling, apoptosis, inflammation, chromatin organization, glycolysis, spliceosome, and rRNA metabolic process. Among these proteins we focused on histones and the 14-3-3 family which are associated to both cell death and cell survival. Confocal microscopy confirmed transfer of histones and 14-3-3 proteins.Protein transfer occurred within neutral lipid vesicles-structures. We also observed that MM cells were inter-connected with a much higher number of neutral lipid structures (nanotubes and bigger structures similar to bags) than in K562 cells (Fig. 1B). Thus, we next analyzed the role of these neutral lipids in MM cell-cell communication. The cholesterol synthesis and lipid transport inhibitor U18666A significantly decreased the number of these lipid connective structures between MM cells, and it was toxic for MM cells. Furthermore, breaking the lipid connection between MM cells with U18666A increased CB-NK cytotoxicity (p<0.05). These effects were not observed in K562 cells. This could be explained by a blockage of the diluent effect mediated by MM cells of the proteins and lipids from CB-NK cells.Last, we analyzed apoptotic levels of 1ºMM cells after co-culture with 2ºMM cells, and the apoptotic levels of the remaining 2ºMM cells. Both populations became early apoptotic, however, after 3-5 days they recovered from early apoptosis, decreasing from 36% of early apoptotic cells to 3%, which was their apoptotic basal levels, and most importantly, without causing cell death, as no difference in the cell number in comparison to control MM cells was observed. Therefore, our data demonstrate that this diluent transmission effect between MM cells might be a new cell mechanism that contributes to tumor cell survival. Importantly, because lipid structures mediate this diluent transmission effect, this tumor cell survival mechanism might be eliminated by using lipid metabolism inhibitors.In conclusion, cell-cell communication increases when MM cells are stressed because of CB-NK presence. Although this causes a transmissible cytotoxicity in a small % of MM cells, a diluent effect also occurs between cells helping MM cells to recover and promoting MM cell survival. Lipid structures play an important role in this cell-cell communication, which may represent a new therapeutic target for MM treatment. DisclosuresNo relevant conflicts of interest to declare.

Noncoding RNA blockade of autophagy is therapeutic in medullary thyroid cancer.

Micro-RNAs are dysregulated in medullary thyroid carcinoma (MTC) and preliminary studies have shown that miRNAs may enact a therapeutic effect through changes in autophagic flux. Our aim was to study the in vitro effect of miR-9-3p on MTC cell viability, autophagy and to investigate the mRNA autophagy gene profile of sporadic versus hereditary MTC. The therapeutic role of miR-9-3p was investigated in vitro using human MTC cell lines (TT and MZ-CRC-1 cells), cell viability assays, and functional mechanism studies with a focus on cell cycle, apoptosis, and autophagy. Post-miR-9-3p transfection mRNA profiling of cell lines was performed using a customized, quantitative RT-PCR gene array card. This card was also run on clinical tumor samples (sporadic: n = 6; hereditary: n = 6) and correlated with clinical data. Mir-9-3p transfection resulted in reduced in vitro cell viability; an effect mediated through autophagy inhibition. This was accompanied by evidence of G2 arrest in the TT cell line and increased apoptosis in both cell lines. Atg5 was validated as a predicted miR-9-3p mRNA target in TT cells. Post-miR-9-3p transfection array studies showed a significant global decline in autophagy gene expression (most notably in PIK3C3, mTOR, and LAMP-1). Autophagy gene mRNAs were generally overexpressed in sporadic (vs. hereditary MTC) and Beclin-1 overexpression was shown to correlate with residual disease. Autophagy is a tumor cell survival mechanism in MTC that when disabled, is of therapeutic advantage. Beclin-1 expression may be a useful prognostic biomarker of aggressive disease.

Identification of secreted proteins that reflect autophagy dynamics within tumor cells

Macroautophagy, a catabolic process of cellular self-digestion, is an important tumor cell survival mechanism and a potential target in antineoplastic therapies. Recent discoveries have implicated autophagy in the cellular secretory process, but potential roles of autophagy-mediated secretion in modifying the tumor microenvironment are poorly understood. Furthermore, efforts to inhibit autophagy in clinical trials have been hampered by suboptimal methods to quantitatively measure tumor autophagy levels. Here, we leveraged the autophagy-based involvement in cellular secretion to identify shed proteins associated with autophagy levels in melanoma. The secretome of low-autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu in physiological 3-dimensional cell culture using quantitative proteomics. These comparisons identified candidate autophagy biomarkers IL1B (interleukin 1, β), CXCL8 (chemokine (C-X-C motif) ligand 8), LIF (leukemia inhibitory factor), FAM3C (family with sequence similarity 3, member C), and DKK3 (dickkopf WNT signaling pathway inhibitor 3) with known roles in inflammation and tumorigenesis, and these proteins were subsequently shown to be elevated in supernatants of an independent panel of high-autophagy melanoma cell lines. Secretion levels of these proteins increased when low-autophagy melanoma cells were treated with the autophagy-inducing tat-BECN1 (Beclin 1) peptide and decreased when ATG7 (autophagy-related 7) was silenced in high-autophagy cells, thereby supporting a mechanistic link between these secreted proteins and autophagy. In addition, serum from metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from patients with low-autophagy tumors. These results suggest that autophagy-related secretion affects the tumor microenvironment and measurement of autophagy-associated secreted proteins in plasma and possibly in tumors can serve as surrogates for intracellular autophagy dynamics in tumor cells.

Abstract A78: Control of tumor cell survival mechanisms by a novel glycan-modifying enzyme

Abstract Altered surface glycosylation was one of the earliest known hallmarks of a tumor cell, however mechanistic insight into the role of glycans in regulating tumor cell phenotype remains limited. Among the subset of glycosyltransferases dysregulated in tumor cells, ST6Gal-I stands out as being consistently upregulated in a variety of different cancers. ST6Gal-I is a Golgi sialyltransferase that adds an a2-6-linked sialic acid, a negatively-charged sugar, to a select group of membrane receptors. Our work has focused on defining ST6Gal-I substrates and elucidating the function of these aberrantly-sialylated receptors in promoting aggressive tumor cell behaviors. Our prior studies showed that ST6Gal-I-directed receptor sialylation enhances ovarian tumor cell invasiveness, and also prevents tumor cell death induced by key apoptotic mediators including galectins, chemotherapy drugs (cisplatin), and certain death receptor activators. In new unpublished studies we find a novel role for ST6Gal-I in controlling downstream signaling from two death receptors, TNFR1 and Fas. Sialylation of Fas and TNFR1 by ST6Gal-I prevents ligand-induced internalization of these receptors, an event essential for apoptotic signaling. However, sialylation of TNFR1 and Fas does not simply shut off receptor activity, but instead diverts signaling toward survival pathways mediated by NFkB and MAPKs. This switch occurs as a consequence of sialylation-dependent retention of TNFR1 and Fas at the cell surface. Given that the ligands for TNFR1 and Fas (TNFa and FasL, respectively) are expressed predominantly by immune cells, we hypothesized that ST6Gal-I sialylation of these receptors may facilitate tumor cell survival within an inflammatory milieu, contributing to escape from immune surveillance. Modeling the tumor microenvironment, we exposed ovarian cancer cells either lacking or overexpressing ST6Gal-I to peritoneal ascites fluid harvested from ovarian cancer patients. Ascites is rich in cytokines including TNFa, sFasL and galectins. Forced expression of ST6Gal-I in ovarian cancer cells conferred protection against ascites-induced cell death, implicating a role for receptor sialylation in tumor cell survival during transcoelomic metastasis. In other unpublished studies, we conducted the first immunohistochemical analyses of ST6Gal-I expression in ovarian cancer, and determined that ST6Gal-I is upregulated in the majority of tumor specimens, including papillary serous and endometrioid carcinomas. Prior to our work, ST6Gal-I had only been measured at the mRNA level in ovarian cancer due to a lack of effective antibodies. To better understand ST6Gal-I's influence on tumor cell phenotype, RNA sequencing/Ingenuity Pathway Analyses were performed in ovarian cancer cells with forced ST6Gal-I overexpression, and it was shown that ST6Gal-I upregulation induced activation of pathways associated with cancer stem cells, including the Wnt signaling axis. These results are consistent with our prior studies in colon tumor cells indicating that ST6Gal-I expression is particularly high in cancer stem cells. Taken together, our results suggest that upregulation of ST6Gal-I in ovarian carcinoma leads to abnormal hypersialylation of a select group of receptors, in turn conferring an invasive, apoptosis-resistant, and chemoresistant tumor cell phenotype. Citation Format: Matthew J. Schultz, Zhongyu Liu, Charles N. Landen, Susan L. Bellis. Control of tumor cell survival mechanisms by a novel glycan-modifying enzyme. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A78.

Human Papillomavirus, p16, and Epidermal Growth Factor Receptor Biomarkers and CT Perfusion Values in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma tumors positive for laboratory biomarkers hrHPV and p16 and negative for EGFR often respond better to nonsurgical organ-preservation therapy than hrHPV-negative, p16-negative, and EGFR overexpressing tumors. CTP has been shown to distinguish which locally advanced head and neck squamous cell carcinomas will respond to induction chemotherapy or chemoradiation. Our purpose was to determine whether a relationship exists between CTP measures and the expression of these laboratory biomarkers, because both appear to separate head and neck squamous cell carcinoma tumors into similar groups. We conducted an institutional review board-approved, Health Insurance Portability and Accountability Act-compliant retrospective review of head and neck CTP in 25 patients with locally advanced head and neck squamous cell carcinoma who had signed informed consent. Eight women and 17 men, 41-80 years of age, constituted a pretreatment group of 18 patients and a palliative group of 7 patients. Tumor biopsy samples were analyzed for overexpression of hrHPV, p16, and EGFR. The hrHPV, p16, and EGFR status of the tumors was correlated with CTP parameters (MTT, BV, BF, CP) by using the Wilcoxon evaluation and Fischer exact test. There were significantly lower CP values in pretreatment tumors overexpressing EGFR (P = .04). CP values ≤17.23 were significantly correlated with EGFR overexpression (P = .015). A trend toward higher CP values was present in hrHPV-positive and p16-overexpressing pretreatment tumors (P = .14). A significant correlation exists between CTP measures and EGFR overexpression in head and neck squamous cell carcinomas, suggesting an association between certain imaging findings and molecular biomarkers. These results may be related to a tumor cell survival mechanism linking perfusion and biomarker expression.

Chemokines and chemokine receptors as promoters of prostate cancer growth and progression.

Prostate cancer (CaP) is estimated to be first in incidence among cancers, with more than 240,000 new cases in 2012 in the United States. Chemokines and their receptors provide survival, proliferation, and invasion characteristics to CaP cells in both primary sites of cancer and metastatic locations. The emerging data demonstrate that many chemokines and their receptors are involved in the multistep process of CaP, leading to metastasis, and, further, that these factors act cooperatively to enhance other mechanisms of tumor cell survival, growth, and metastasis. Changes of chemokine receptor cohorts may be necessary to activate tumor-promoting signals. Chemokine receptors can activate downstream effectors, such as mitogen-activated protein kinases, by complex mechanisms of ligand-dependent activation of cryptic growth factors; guanosine triphosphate-binding, protein-coupled activation of survival kinases; or transactivation of other receptors such as ErbB family members. We describe vanguard research in which more than the classic view of chemokine receptor biology was clarified. Control of chemokines and inhibition of their receptor activation may add critical tools to reduce tumor growth, especially in chemo-hormonal refractory CaP that is both currently incurable and the most aggressive form of the disease, accounting for most of the more than 28,000 annual deaths.

Abstract 3772: The amplified cancer gene LAPTM4B plays a critical role in autophagosome maturation and promotes cancer cell survival during metabolic stress

Abstract Research has shown that lysosome-mediated autophagy is up regulated in response to various stimuli such as hypoxia, nutrient limitation, and drug-induced cytotoxic stress. In cancer progression, autophagy plays a cytoprotective role by generating needed nutrients or by eliminating damaged cellular components. Although autophagy has been recognized as a critical tumor cell survival mechanism and much is known about the induction and early steps of autophagy, the molecule(s) which control the later stages of autophagic progression are less clear. Previously, we have identified lysosomal-associated protein transmembrane 4b (LAPTM4B) as an amplified and over-expressed gene in breast cancer that is associated with de novo resistance to anthracycline chemotherapy and increased risk of distant metastatic recurrence. We reported that LAPTM4B overexpression alters intracellular trafficking of anthracyclines by retaining the drug in the cytoplasmic compartment thereby limiting drug-induced DNA damage in the nucleus. However, how this protein contributes to tumor cell survival in the setting of other types of stress requires further investigation. In the present studies, we used fluorescence microscopy to confirm LAPTM4B protein distribution in the lysosomal compartment of cells. Depletion of this gene results in increased lysosome membrane permeability as demonstrated by release from lysosomes of low to intermediate molecular weight dextran. Furthermore, we find that over expression of LAPTM4B renders cells resistant to apoptosis when placed in nutrient-free media suggesting a possible role of LAPTM4B in promoting lysosome-mediated autophagy. We induced autophagy by serum starvation in BT549 breast cancer cells with or without depletion of LAPTM4B. Autophagic progression was monitored by immunofluorescence for the autophagosome marker, LC3, and for degradation of P62 as evidence of autolysosome maturation. We find that depletion of LAPTM4B interrupted autophagosome maturation to autolysosomes as indicated by appearance of LC3 but absence of P62 degradation. In addition, we observed aggregation of autophagosomes in LAPTM4B-depleted cells and caspase activation, indicative of apoptosis. These events are a recognized sequela of blocked autophagic maturation. In summary, we find that ablation of LAPTM4B results in increased lysosome membrane permeability, inhibition of autophagosome-lysosome fusion, and toxic accumulation of autophagasomes triggering cell death. These results suggest a novel function for the amplified cancer gene, LAPTM4B, in promoting the later stages of autophagic maturation and tumor resistance to apoptosis-inducing stress. Targeting LAPTM4B may provide a new strategy to prevent metabolic adaptation of cancer cells by abolishing the cytoprotective effects of autophagy and increasing drug therapy efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3772. doi:10.1158/1538-7445.AM2011-3772

Abstract SY04-02: Environmental stress and breast cancer biology: What is the link

Abstract For many decades, identifying neuroendocrine mechanisms linking psychosocial factors (e.g., chronic stressors) to human disease has been the subject of intense interest by both social and biological scientists. Together, the endocrine and autonomic nervous systems comprise an individual's physiological response to both chronic and acute social stressors; these systems also mediate signaling pathways and gene expression changes at the cellular level that have the potential to alter tumor biology. However, it is only recently that the detailed molecular components connecting biopsychological stressors and cancer biology have begun to be uncovered. For example, in preclinical models of both breast and ovarian cancer, there is growing evidence linking biopsychological stressors and ensuing changes in neuroendocrine dynamics to the promotion of tumor growth. Both the glucocorticoid stress hormones and the adrenergic systems can induce tumor cell survival mechanisms and alter cell proliferation, tumor invasion, and angiogenesis. In addition, these signals affect inflammatory and metabolic tissues that modulate tumor growth through systemic and microenvironmental effects. Assessing the precise mechanisms of these interactions requires an appreciation of the subtleties of measuring the social environment, the individual's behavioral and neuroendocrine response, as well as the tumor and its environment. Our collaborative development of a model of social isolation as it pertains to a transgenic model of human breast cancer (SV40Tag) and a Sprague-Dawley female rat model of spontaneous mammary gland cancer will be presented. In both models, we found that chronic social isolation reduces exploratory behavior in a novel environment as well as heightening the corticosterone response to an acute restraint stressor. In the SV40 Tag model, we examined gene expression differences in the mammary glands of isolated versus grouped female mice prior to invasive tumor development and uncovered a significant increase in lipid synthesis and glycolytic pathway genes, suggesting an association of the social environment with cancer-promoting metabolic changes. Interestingly, many of these changes in gene expression appear most significant in the adipocyte fraction of the mammary gland. For example, the increased expression of the key metabolic genes Acaca, Hk2, and Acly, initially observed in the whole mammary gland, was found to be significantly elevated only in the adipocyte fraction. Furthermore, metabolic gene expression was not consistently increased in the visceral fat of social isolates. These results suggest that exposure to chronic social isolation results in mammary fat depot-specific metabolic gene expression changes, followed by increased growth of invasive tumors. The implications and limitations of these two breast cancer models – one transgenic and one spontaneous – will be discussed in the context of understanding specific mechanisms that confer increased nonestrogen-dependent mammary tumor growth in association with chronic exposure to a psychosocial stressor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY04-02. doi:10.1158/1538-7445.AM2011-SY04-02

A Novel In Vitro Model of Lymphatic Metastasis from Colorectal Cancer

For many types of cancers, successful metastasis is critically dependent on tumor cell survival under flow conditions in the lymphatic system as well as attachment to the lymphatic endothelium at distal sites. In the lymphatic system, tumor cells are exposed to dynamic forces of laminar shear stress; however, there are currently no models to study the effects of these dynamic fluid forces on colorectal cancer metastasis. This study aims to establish the rudiments of an in vitro flow system that mimics the conditions to which tumor colorectal cancer cells (CRCCs) are exposed during lymphatic spread. Human CRCCs (RKO and HCT-8) were cultured on collagen-1 coated glass slides in 10% fetal bovine serum, and grown until 50% confluence under static conditions. Subconfluent cells were then treated with laminar shear stress (1.2 dynes/cm(2)) using a Flexcell Streamer (Flexcell International Corp., Hillsborough, NC) parallel plate chamber for up to 48 h, in the continued presence of serum. Control conditions consisted of cells maintained under static conditions (0 dynes/cm(2)). Cells were examined with digital microscopy. Cell number was determined directly by cell count. Poly (ADP-ribose) polymerase-1, caspase-3, matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor C levels were measured by Western blot. CRCCs survived under conditions of lymphatic flow (1.2 dynes/cm(2)), and were confluent by 48 h. Although a small number of cells (10% to 15%) initially detached upon exposure to shear stress, the majority of cells remained attached, and mitotic cells were observed. Cells demonstrated increased attachment and spreading under lymphatic flow compared with cells kept under control conditions. Cell number increased in cells treated with both lymphatic flow and static conditions by similar amounts until confluence was achieved. Cleaved products of poly (ADP-ribose) polymerase-1 and caspase-3 were not observed. MMP-2, MMP-9, and vascular endothelial growth factor C were expressed to similar degrees at all time points in cells exposed to lymphatic flow. Using a novel in vitro model of lymphatic flow, we describe colorectal tumor cell proliferation and expression of peptides critical to lymphatic spread under flow conditions. The ability to model lymphatic spread in vitro will allow additional studies to determine mechanisms of tumor cell survival in the lymphatic system.

Screening and identification of differentially expressed transcripts in circulating cells of prostate cancer patients using suppression subtractive hybridization

BackgroundTumor metastasis and changes in host immunosurveillance are important components in cancer development. Tumor cell invasion into the bloodstream is an essential step for systemic metastasis. Currently, the detection of tumor cells in the circulation is mainly dependent upon the utilization of known epithelial cell markers. However, expression of these molecules is not limited to cancer patients; healthy people also have a small number of epithelial cells in their circulation. Utilizing these markers to detect circulating tumor cells (CTCs) cannot adequately explain the mechanisms of tumor cell survival or their development of metastatic potential in peripheral blood. The immune system can also evolve along with the cancer, actually promoting or selecting the outgrowth of tumor variants. Unfortunately, both metastasis and immunosurveillance remain mysterious and are debatable because we have yet to define the molecules that participate in these processes. We are interested in identifying the existence of expressed genes, or mRNA species, that are specifically associated with circulating cells of cancer-bearing patients using prostate cancer (PCa) as a model.ResultsWe established two comprehensive subtracted cDNA libraries using a molecular technique called suppression subtractive hybridization. This technique selectively amplifies transcripts that are specifically expressed in circulating cells of either PCa patients or healthy men. Following sequencing reaction, we showed that 17 out of 23 (73.9%) sequenced clones did not match any mRNAs in the GenBank database. This result suggests that genes associated with alterations in circulating cells of cancer-bearing patients are largely unknown. Semi-quantitative RT-PCR confirmed that two genes are up-regulated in circulating cells of PCa patients, whereas another two genes are down-regulated in the same patients.ConclusionThe comprehensive gene expression analysis is capable of identifying differentially expressed genes in circulating cells of healthy men and PCa patients. We did not attempt to enrich specific cell types in this study because phenotypes of CTCs and subsets of leukocytes participating in immunosurveillance remain largely unknown. Continuous studies of these differentially expressed genes will eventually lead us to understand the mechanisms involved in tumor metastasis and immune modulation during cancer development.

Role of gamma-glutamyltransferase in the homeostasis of glutathione during oxidative and nitrosative stress.

Glutathione (GSH) is an important and abundant antioxidant in most cells, including cancer cells. The GSH level can be significantly reduced during oxidative and nitrosative stress, and GSH depletion is an early step in apoptosis. As most cells have no uptake mechanism of GSH, they depend on resynthesis to prevent cell death. A limiting factor in cellular GSH synthesis is the availability of cyst(e)ine (for recent reviews, see [1,2]). During tumor progression cancer cells tend to develop invasive and metastatic growth and resistance to therapy. They also show metabolic alterations that are termed loss of differentiation, and frequently these changes include increased resistance towards oxidative and nitrosative stress [3,4]. It has been shown that GSH protects tumor cells against oxidative stress in liver microvasculature [5] and that tumor cells with elevated GSH or ability to resynthesise GSH possess a higher invasive potential [4,6]. NO is highly tumoricidal in the presence of H2O2 [6,7], and these agents appear to be part of a natural defence towards metastatic growth [8]. GSH plays an important role in the protection of tumor cells against NO mediated apoptosis [9,10] and intracellular GSH will therefore contribute to the mechanisms of tumor cell survival during metastatic growth. The enzyme γ-glutamyltransferase (GGT) initiates the breakdown of extracellular glutathione by hydrolysing the γ-glu-cys bond in GSH. The remaining part, cys-glu, is then hydrolysed by peptidases. Cellular uptake of the amino acids will thus provide the monomers needed for GSH biosynthesis [11] and it has been suggested that GGT has significant importance for GSH homoestasis when the level of cyst(e)ine is reduced [12–14]. Fibroblasts requiring cysteine can grow in cysteine-free medium if supplemented with GSH [12,15]. Cells cultivated in vitro have access to surplus amounts of cystine

Molecular Mechanisms of Tumor Cell Survival, Growth, and Apoptosis During Organ-Specific Metastasis

Metastasis is a highly selective nonrandom process favoring the survival of minor subpopulations of metatastatic cells that preexist within the primary tumor. The cellular and molecular mechanisms that regulate the metastasis of tumor cells to specific organs are diverse and both tumor and organ-specific (1). Many examples exist in which malignant tumors metastasize to specific organs. As Paget proposed in 1889 (2), and as our recent biological and molecular evidence demonstrates, the organ microenvironment influences the invasion, survival, growth, and apoptosis of particular tumor cells. This hypothesis explains metastatic colonization patterns that cannot be due to solely mechanical lodgement/anatomical considerations (1). Successful metastasis therefore involves the interaction of tumor cells with a compatible milieu provided by a particular organ environment.Recent experimental evidence suggests that paracrine stimulation of tumor cells by organ-derived growth factors and cytokines is one mechanism which determines the target organ preference of disseminated cancer cells.