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Complete list of proteins identified in PEAK1-depleted PDAC cells by LC/MS/MS

Western blots showing PEAk1 and YAP1 depletion in PDAC cells treated with two independent shRNAs

In pancreatic ductal adenocarcinoma (PDAC), mutant KRAS stimulates the translation initiation factor eIF5A and upregulates the focal adhesion kinase PEAK1, which transmits integrin and growth factor signals mediated by the tumor microenvironment. Although eIF5A-PEAK1 signaling contributes to multiple aggressive cancer cell phenotypes, the downstream signaling processes that mediate these responses are uncharacterized. Through proteomics and informatic analyses of PEAK1-depleted PDAC cells, we defined protein translation, cytoskeleton organization, and cell-cycle regulatory pathways as major pathways controlled by PEAK1. Biochemical and functional studies revealed that the transcription factors YAP1 and TAZ are key targets of eIF5A-PEAK1 signaling. YAP1/TAZ coimmunoprecipitated with PEAK1. Interfering with eIF5A-PEAK1 signaling in PDAC cells inhibited YAP/TAZ protein expression, decreasing expression of stem cell–associated transcription factors (STF) including Oct4, Nanog, c-Myc, and TEAD, thereby decreasing three-dimensional (3D) tumor sphere growth. Conversely, amplified eIF5A-PEAK1 signaling increased YAP1/TAZ expression, increasing expression of STF and enhancing 3D tumor sphere growth. Informatic interrogation of mRNA sequence databases revealed upregulation of the eIF5A-PEAK1-YAP1-TEAD signaling module in PDAC patients. Taken together, our findings indicate that eIF5A-PEAK1-YAP signaling contributes to PDAC development by regulating an STF program associated with increased tumorigenicity. Cancer Res; 77(8); 1997–2007. ©2017 AACR.

Accumulating evidence indicates that obesity with its associated metabolic dysregulation, including hyperinsulinemia and aberrant circadian rhythms, increases the risk for a variety of cancers including postmenopausal breast cancer. Caloric restriction can ameliorate the harmful metabolic effects of obesity and inhibit cancer progression but is difficult to implement and maintain outside of the clinic. In this study, we aim to test a time-restricted feeding (TRF) approach on mouse models of obesity-driven postmenopausal breast cancer. We show that TRF abrogates the obesity-enhanced mammary tumor growth in two orthotopic models in the absence of calorie restriction or weight loss. TRF also reduces breast cancer metastasis to the lung. Furthermore, TRF delays tumor initiation in a transgenic model of mammary tumorigenesis prior to the onset of obesity. Notably, TRF increases whole-body insulin sensitivity, reduces hyperinsulinemia, restores diurnal gene expression rhythms in the tumor, and attenuates tumor growth and insulin signaling. Importantly, inhibition of insulin secretion with diazoxide mimics TRF whereas artificial elevation of insulin through insulin pumps implantation reverses the effect of TRF, suggesting that TRF acts through modulating hyperinsulinemia. Our data suggest that TRF is likely to be effective in breast cancer prevention and therapy., Obesity and its associated metabolic changes, including hyperinsulinemia and aberrant circadian rhythms, increases the risk for a variety of cancers including postmenopausal breast cancer. Here, the authors show that restricting when mice eat, but not what or how much they eat, delays breast cancer initiation and reduces tumor growth in obese mice in addition to improving insulin sensitivity and restoring circadian rhythms.

SUMMARYSignaling networks balance the activities of many physically interacting proteins and perturbations to this network influence downstream signaling, potentially leading to oncogenic states. Using affinity purification-mass spectrometry we defined this network for all 90 human tyrosine kinases revealing 1,463 mostly novel interactions between these key cancer proteins and diverse molecular complexes. Modulation of interactor levels altered growth phenotypes associated with corresponding tyrosine kinase partners suggesting that tumors may alter the stoichiometries of interactors to maximize oncogenic signaling. We show that the levels of EGFR interactors delineates this form of network oncogenesis in 19% of EGFR wild-type lung cancer patients which were mostly otherwise oncogene negative, predicting sensitivity to EGFR inhibitors in vitro and in vivo. EGFR network oncogenesis occurs through mechanistically distinct network alleles often in cooperation with weak oncogenes in the MAPK pathway. Network oncogenesis may be a common and targetable convergent mechanism of oncogenic pathway activation in cancer.HIGHLIGHTSA human tyrosine kinome protein interaction map reveals novel physical and functional associations.Dependence on oncogenic tyrosine kinases is modulated through perturbation of their interactors.EGFR network oncogenesis in up to 19% of EGFR wild-type lung cancers is targetable.EGFR network oncogenesis cooperates with weak oncogenes in the MAPK pathway.

Background: The prevalence of obesity and the metabolic syndrome (MetS) has increased dramatically in developed countries over the last three decades (Flegal et al., 2012). Numerous studies indicate that adiposity and the MetS are independent risk factors for multiple diseases including cancer, particularly postmenopausal breast cancer (Kim et al., 2018). Therefore improving the metabolic health of obese postmenopausal women may mitigate their risk for breast cancer. Accumulating evidence suggests that time-restricted feeding (TRF), a form of intermittent fasting in which food intake is limited to a defined period during the normal active phase, can have a positive influence on metabolic health. Importantly, interventional studies in obese mice and small clinical studies in humans have demonstrated that TRF can improve metabolic health even though obesity is maintained (Sutton et al., 2018). Time restriction rather than calorie restriction is thus a promising method to control the negative sequelae of obesity, due to the hunger and irritability that reduces compliance with long-term calorie restriction. The objective of this study was to investigate whether TRF attenuates breast cancer in a mouse model of postmenopausal obesity and whether this effect is mediated by reducing the hyperinsulinemia associated with obesity. Methods: Ovariectomized mice were used as postmenopausal mice model. The ovariectomized mice were initially made obese by feeding 60% high fat diet (HFD) for 10 weeks and then grouped into a continued ad libitum group (24 h access to food) or a TRF group (8 h access to food during active phase). For an orthotopic tumor model, mice were injected with E0771 breast cancer cells into four mammary fat pads per mouse three weeks following the start of TRF. As a tumor initiation model, transgenic PyMT mice were used to assess tumor onset and growth following the same TRF or AL access to the HFD. The insulin dependency of tumor growth was studied by increasing insulinemia using an implanted insulin pump, or by reducing insulin secretion using diazoxide. Insulin effects on tumor cell proliferation and migration was further validated in vitro. Results and Conclusion: TRF had a dramatic effect, reducing tumor growth in obese mice fed a high fat diet (HFD) to levels seen in lean mice. Tumor growth and initiation was also delayed in the transgenic PyMT model of mammary tumorigenesis. Our results further suggest that the antitumor effect of TRF is at least partially mediated by reducing hyperinsulinemia, suggesting that this intervention may be effective in breast cancer prevention and therapy. References: • Flegal, K. M. et al., (2012), JAMA 307, 491-497. • Kim, N. H. et al. (2018). Dig Dis Sci 63, 3126-3133. • Sutton, E. F. et al. (2018). Cell Metab 27, 1212-1221 e1213.

The cancer stem cell (CSC) paradigm posits that specific cells within a tumor, so-called CSC-like cells, have differing levels of tumorigenicity and chemoresistance. Original studies of CSCs identified them in human cancers and utilized mouse xenograft models to define the cancer initiating properties of these cells, thereby hampering the understanding of how immunity could affect CSCs. Indeed, few studies have characterized CSCs in the context of cancer immunoediting, and it is currently not clear how immunity could impact on the levels or stem-like behavior of CSCs. Using the well-studied 3′methylcholanthrene (MCA) model of primary sarcoma formation, we have defined a CSC-like population within MCA-induced sarcomas as expressing high levels of stem cell antigen-1 (Sca-1) and low levels of CD90. These Sca-1(+)CD90(−) CSC-like cells had higher tumor initiating ability, could spontaneously give rise to Sca-1-negative cells, and formed more sarcospheres than corresponding non-CSC-like cells. Moreover, when examining MCA-induced sarcomas that were in the equilibrium phase of cancer growth, higher levels of CSC-like cells were found compared to MCA-induced sarcomas in the escape phase of cancer progression. Notably, CSC-like cells also emerged during escape from anti-PD-1 or anti-CTLA4 therapy, thus suggesting that CSC-like cells could evade immune therapy. Finally, we demonstrate that paradoxically, interferon (IFN)-γ produced in vivo by immune cells could promote the emergence of CSC-like cells. Our findings define the existence of a Sca1(+)CD90(−) CSC-like population in the MCA-sarcoma model capable of differentiation, tumorsphere formation, and increased tumor initiation in vivo. These cells may also act as mediators of immune resistance during cancer immunoediting and immune therapy.

The impact of volatile anesthetics on cancer progression has been observed for decades, but sex differences have not been described. Male and female immune systems vary considerably, and the immune system plays an important role in limiting cancer growth. Currently, mouse models describing the impact of volatile anesthetics on cancer growth are limited to same-sex models. In this brief report, we describe a sex-specific impact of isoflurane on melanoma growth observed in wild-type but not in immune-deficient mice. Future experimental designs related to anesthesia and cancer should evaluate the biological variable of sex in a systematic manner.

In pancreatic ductal adenocarcinoma (PDAC), mutant KRAS stimulates the translation initiation factor eIF5A and upregulates the focal adhesion kinase PEAK1, which transmits integrin and growth factor signals mediated by the tumor microenvironment. Although eIF5A-PEAK1 signaling contributes to multiple aggressive cancer cell phenotypes, the downstream signaling processes that mediate these responses are uncharacterized. Through proteomics and informatic analyses of PEAK1-depleted PDAC cells, we defined protein translation, cytoskeleton organization, and cell-cycle regulatory pathways as major pathways controlled by PEAK1. Biochemical and functional studies revealed that the transcription factors YAP1 and TAZ are key targets of eIF5A-PEAK1 signaling. YAP1/TAZ coimmunoprecipitated with PEAK1. Interfering with eIF5A-PEAK1 signaling in PDAC cells inhibited YAP/TAZ protein expression, decreasing expression of stem cell–associated transcription factors (STF) including Oct4, Nanog, c-Myc, and TEAD, thereby decreasing three-dimensional (3D) tumor sphere growth. Conversely, amplified eIF5A-PEAK1 signaling increased YAP1/TAZ expression, increasing expression of STF and enhancing 3D tumor sphere growth. Informatic interrogation of mRNA sequence databases revealed upregulation of the eIF5A-PEAK1-YAP1-TEAD signaling module in PDAC patients. Taken together, our findings indicate that eIF5A-PEAK1-YAP signaling contributes to PDAC development by regulating an STF program associated with increased tumorigenicity. Cancer Res; 77(8); 1997–2007. ©2017 AACR.

Evidence of cancer immunosurveillance and immunoediting processes has been primarily demonstrated in mouse models of chemically induced oncogenesis. Although these models are very tractable, they are characterized by high mutational loads that represent a minority of human cancers. In this study, we sought to determine whether cancer immunosurveillance and immunoediting could be demonstrated in a more clinically relevant oncogene-induced model of carcinogenesis, the MMTV-PyMT (PyMT) mammary carcinoma model. This model system in the FVB/NJ strain background was previously used to demonstrate that adaptive immunity had no role in limiting primary cancer formation and in fact promoted metastasis, thus calling into question whether cancer immunosurveillance operated in preventing the development of breast cancer. Our current study in the C57BL/6 strain backgrounds provides a different conclusion, as we report here the existence of an adaptive immunosurveillance of PyMT mammary carcinomas using two independent models of immune deficiency. PyMT mice bred onto a Rag1−/− background or immune suppressed by chronic tacrolimus therapy both demonstrated accelerated development of mammary carcinomas. By generating a bank of cell lines from these animals, we further show that a subset of PyMT cell lines had delayed growth after transplantation into wild-type (WT) syngeneic, but not immune-deficient hosts. This reduced growth rate in immunocompetent animals was characterized by an increase in immune cell infiltration and tissue differentiation. Furthermore, loss of the immune cell infiltration that characterized immunoediting of slow growing cell lines, changed them into fast growing variants capable of progressing in the immunocompetent model. In conclusion, our study provides evidence that immunosurveillance and immunoediting of PyMT-derived cell lines modulate tumor progression in this oncogene-induced model of cancer.

SummaryThe process of cancer immunoediting generates a repertoire of cancer cells that can persist in immune-competent hosts. In its most complex form, this process begins with the elimination of highly immunogenic unedited tumor cells followed by the escape of less immunogenic edited cells. Although edited tumors can release immunosuppressive factors, it is unknown whether unedited tumors produce cytokines that enhance antitumor function. Utilizing gene microarray analysis, we found the cytokine interleukin 17D (IL-17D) was highly expressed in certain unedited tumors but not in edited mouse tumor cell lines. Moreover, forced expression of IL-17D in edited tumor cells induced rejection by stimulating MCP-1 production from tumor endothelial cells, leading to the recruitment of natural killer (NK) cells. NK cells promoted M1 macrophage development and adaptive immune responses. IL-17D expression was also decreased in certain high-grade and metastatic human tumors, suggesting that it can be targeted for tumor immune therapy.

Cancer immunosurveillance eradicates certain neoplasms, but the selective pressure exerted by this active surveillance leads to the emergence of immune evasive tumor clones in a process called cancer immunoediting. Natural killer (NK) cells are potent effectors of cancer immunoediting and can destroy tumors directly via exocytosis of cytotoxic granules or indirectly by producing interferon γ to activate M1 and TH1 immune responses. This review gathers current knowledge of NK immunosurveillance of primary tumors induced in mice and highlights the importance of NK immunosurveillance for human cancers. Evidence of NK immunoediting, as revealed by studies using NK-deficient models, demonstrates how exposure to NK cells engenders modification of cancer immunogenicity to permit survival and progression of the tumor clone in an immunocompetent environment.

Despite clinical potential and recent advances, durable immunotherapeutic ablation of solid tumors is not routinely achieved. IL15 expands natural killer cell (NK), natural killer T cell (NKT) and CD8+ T-cell numbers and engages the cytotoxic program, and thus is under evaluation for potentiation of cancer immunotherapy. We found that short-term therapy with IL15 bound to soluble IL15 receptor α–Fc (IL15cx; a form of IL15 with increased half-life and activity) was ineffective in the treatment of autochthonous PyMT murine mammary tumors, despite abundant CD8+ T-cell infiltration. Probing of this poor responsiveness revealed that IL15cx only weakly activated intratumoral CD8+ T cells, even though cells in the lung and spleen were activated and dramatically expanded. Tumor-infiltrating CD8+ T cells exhibited cell-extrinsic and cell-intrinsic resistance to IL15. Our data showed that in the case of persistent viral or tumor antigen, single-agent systemic IL15cx treatment primarily expanded antigen-irrelevant or extratumoral CD8+ T cells. We identified exhaustion, tissue-resident memory, and tumor-specific molecules expressed in tumor-infiltrating CD8+ T cells, which may allow therapeutic targeting or programming of specific subsets to evade loss of function and cytokine resistance, and, in turn, increase the efficacy of IL2/15 adjuvant cytokine therapy. Cancer Immunol Res; 4(9); 799–811. ©2016 AACR.

Background: Abundant evidence indicates that obesity increases risk for breast cancer and the incidence further increases by 40% in obese postmenopausal women. Therefore it is envisioned by diverse research groups that the risk of breast cancer in obese postmenopausal women is an ever-increasing menace that needs to be curbed soon with effective practical strategies that could have a far-reaching impact. In this setting, time-restricted feeding (TRF), the practice of consuming ad libitum energy during the normal active phase, has been demonstrated to reinforce normal metabolic regulation, thereby attenuating obesity-driven metabolic deregulation. Although studies have assessed the effect of TRF on metabolism, no investigations have been carried out in human or mouse exploring TRF in cancer remission. Therefore, the present work is to understand the efficacy of TRF for improved breast cancer remission in postmenopausal obese female mice. Further, the work explores the mechanistic link of TRF for reduced tumor growth. Methods: Ovariectomized and 4-vinylcyclohexene diepoxide (VCD) treated mice were used as a postmenopausal model. Both ovariectomized and VCD mice were made obese by feeding them with 60% high-fat diet (HFD) and then grouped into ad libitum group (24 h access to food) and TRF group (8 hr access to food at night) to assess metabolic and tumor growth effect of TRF. To develop breast tumor, mice were injected with py230 breast cancer cell line into the mammary fat pad. The metabolic effect of TRF was assessed by glucose tolerance test, insulin tolerance test, body weight measurement, food intake, lipid accumulation in liver by HE staining and measurement of different tissue weight. Measuring the tumor volume over time and tumor weight assessed TRF effect on tumor growth. Performing a tumor growth study in mice fed with HFD and HFD containing diazoxide assessed insulin dependency of tumor growth in ad libitum group. Insulin-dependent tumor growth was validated by tumor growth study in normal chow-fed mice implanted with insulin pump or without pump (control). Results: The preliminary studies in ovariectomized and VCD-treated postmenopausal mice suggest that restricting access to Western-style HFD in active night phase improves insulin resistance, glucose tolerance, and hepatic steatosis. Further, TRF exhibited reduced tumor growth compared to ad libitum group. More importantly, the results from tumor growth study in mice fed with HFD with/without diazoxide or normal chow mice with/without insulin pump, suggest that the tumor growth is insulin dependent and TRF may be acting through attenuating insulin signaling. Conclusion: Experimental and animal model data corroborate that TRF improves metabolic deregulation and reduces breast tumor growth in HFD-fed obese mice. The results suggest putative application of such therapeutic intervention for breast cancer therapy. Citation Format: Manasi Das, Emilie Gross, Deepak Kumar, Consuelo Sauceda, Hyun-Tae Park, Dorothy D. Sears, Lesley Ellies, Nicholas Webster. Time-restricted feeding: A dietary intervention to treat breast cancer in postmenopausal obese mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 377.

Cells undergoing xenobiotic or oxidative stress activate the transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2), which initiates an intrinsic "stress surveillance" pathway. We recently found that the cytokine IL-17D effects a form of extrinsic stress surveillance by inducing antitumor immunity, but how IL-17D is regulated remains unknown. Here, we show that Nrf2 induced IL-17D in cancer cell lines. Moreover, both Nrf2 and IL-17D were induced in primary tumors as well as during viral infection invivo. Expression of IL-17D in tumors and virally infected cells is essential for optimal protection of the host as il17d(-/-) mice experienced a higher incidence of tumors and exacerbated viral infections compared to wild-type (WT) animals. Moreover, activating Nrf2 to induce IL-17D in established tumors led to natural killer cell-dependent tumor regression. These data demonstrate that Nrf2 can initiate both intrinsic and extrinsic stress surveillance pathways and highlight the use of Nrf2 agonists as immune therapies for cancer and infection.

Rituximab (RTX, anti-CD20 antibody) combined with chemotherapy is currently standard treatment for chronic lymphocytic leukemia (CLL). Serum level of IL-8 is a prognostic factor for CLL that correlates with disease stage. We investigated whether endogenous IL-8 affects RTX or Obinutuzumab (GA101) B-leukemic depletion mediated by natural killers (NK). Using whole peripheral blood lymphocytes from untreated CLL patients, RTX, but most significantly GA101, were effective in B-cell depletion and NK activation. IL-8 inhibition completely inhibited B-cell depletion by RTX and reduced GA101-induced B-cell depletion. Altogether results underline IL-8 as an endogenous NK co-activator and confirm GA101 therapeutic potential for CLL treatment.

The concept of cancer stem cells has revolutionized our current vision of cancer development and was validated in solid tumors and cancers of the primitive hematopoietic compartment. Proof of the principle is still lacking, however, in malignancies of differentiated B-cells. We review here the current literature, which nevertheless suggests hierarchical organizations of the tumor clone for mostly incurable B-cell cancers such as multiple myeloma, lymphomas and B-chronic lymphocytic leukemia. We propose two models accounting for cancer stem cells in these contexts: a “top-to-bottom” clonal hierarchy from memory B-cells and a “bottom-to-top” model of clonal reprogramming. Selection pressure on the growing tumor can drive such reprogramming and increase its genetic diversity.

B-cell chronic lymphocytic leukemia (B-CLL) therapy remains unsatisfactory due to repeated resurgences of the chemoresistant disease. In this study, we investigated the basis of this chemoresistance by applying the 'side population' (SP) analysis to blood samples from B-CLL patients. We report the existence of few natural SP cells, which harbors phenotypic and cytogenetic hallmarks of B-CLL in most patients with this disease (n=22). SP cells appeared resistant to conventional B-CLL treatments, such as Fludarabine, Bendamustin or Rituximab. Indeed, treatment with Fludarabine (16/18 cases) or Bendamustin (5/7 cases) resulted in complete elimination of non-SP, whereas cells displaying the SP phenotype were the only surviving. Although some B-CLL SP cells were innately chemoresistant, chemotherapy by Fludarabine selected not only innate SP cells but also induced some acquired SP cells, which arose from non-SP by drug-driven evolution. This SP selection by chemotherapeutic treatments is further supported by the overall increase of the SP percentage in patients who experienced chemotherapy in the preceding year. Functionally, proliferative stimulation of SP cells was able to partially replenish in vitro the non-SP cell compartment of the B-CLL disease. The chemoresistance of B-CLL relies, in our model, on the cellular heterogeneity of B-CLL SP cells and on their regenerating dynamics.

Thirty B-cell chronic lymphocytic leukemia patients were treated with fludarabine-cyclophosphamide-rituximab (FCR) and immune cell counts (natural killer (NK) cells, CD4, CD8, Tgammadelta and monocytes) were monitored from the end of treatment (EOT) up to 36 months (M36). Moreover, nonleukemic peripheral blood lymphocyte cytotoxicity (PBL/CTC) as well as rituximab (RTX)-dependent PBL/CTC was also measured at the initiation of therapy, EOT and M12. These parameters were correlated with post-FCR monitoring of the minimal residual disease (MRD) level in blood using a four-color flow cytometry technique. FCR induced a profound and sustained depletion of all T-cell populations, Tgammadelta being the most affected, whereas NK cells were relatively preserved. Both basal and interleukin-2-stimulated nonleukemic PBL/CTC against MEC-2, a CLL cell line, increased during the post-FCR period. There was no correlation between immune recovery parameters and MRD progression profile, except that patients with high post-FCR CD4(+) counts experienced rapid MRD progression. MRD at M12 predicts clinical relapse. The limited data show RTX-mediated LBL/CTC activity against autologous B-cell cells in individuals with1% residual disease at M12, opening avenues for immunomodulation post-FCR with anti-CD20 antibodies. To conclude, our study suggests that MRD increase at M12 precedes disease evolution post-FCR, and should be assessed as a surrogate marker for proactive management of CLL relapse.

Rituximab (RTX), a monoclonal antibody directed against the CD20 protein, is a drug commonly used in the treatment of B-cell–derived lymphoid neoplasias and of antibody-mediated autoimmune diseases. In addition to cell- and complement-mediated B-cell depletion, RTX is thought to inhibit B-cell survival and proliferation through negative regulation of canonical signaling pathways involving Akt, ERK, and mammalian target of rapamycin. However, surprisingly, although B-cell receptor (BCR) signaling has been considered critical for normal and more recently, for neoplastic B cells, the hypothesis that RTX could target BCR has never been investigated. Using follicular lymphoma cell lines as models, as well as normal B cells, we show here, for the first time, that pretreatment with RTX results in a time-dependent inhibition of the BCR-signaling cascade involving Lyn, Syk, PLCγ2, Akt, and ERK, and calcium mobilization. The inhibitory effect of RTX correlates with decrease of raft-associated cholesterol, complete inhibition of BCR relocalization into lipid raft microdomains, and down-regulation of BCR immunoglobulin expression. Thus, RTX-mediated alteration of BCR expression, dynamics, and signaling might contribute to the immunosuppressive activity of the drug.

The development of therapeutic monoclonal antibodies (mAbs) has revolutionized the treatment of cancer along the last ten years. The best examples of their therapeutic efficacies have been obtained with rituximab for the treatment of CD20+ B-cell Non-Hodgkin Lymphoma (B-NHL), and several others antibodies with optimized bioactivities are now being developed for the treatment of various malignant hemopathies. We review here the main drugs developed in this field, and present some emerging concepts able to improve the bioactivities of the next generation of therapeutic mAbs.

In human blood, 1% to 5% of lymphocytes are gammadelta T cells; they mostly express the gammadelta T-cell receptor (TCR)Vgamma9, recognize nonpeptide phosphoantigens (PAgs) produced by microbes and tumor cells, and mediate different modes of lytic activities directed against tumor target cells. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by cytolytic lymphoid cells is essential for the clinical activity of anticancer monoclonal antibodies (mAbs), but whether PAgs affect ADCC by gammadelta T cells is unknown. Here we report that, in association with the CD20(+)-specific mAb rituximab (RTX), the synthetic PAg bromohydrin pyrophosphate (BrHPP) increased TCRVgamma9(+) cell binding to CD20(+) lymphoma cells in vitro. This combination activated phospho-ZAP70 and phospho-ERK1/2 signaling in TCRVgamma9(+) cells and strongly enhanced their ADCC activity. We obtained similar results with BrHPP in the context of the mAbs alemtuzumab and trastuzumab. Furthermore, BrHPP enhanced RTX-mediated depletion of CD20(+) cells in vitro from peripheral blood mononuclear cells of healthy subjects and enhanced ADCC by gammadelta T cells from patients with chronic lymphocytic leukemia. In cynomolgus macaques, a regimen combining RTX, BrHPP, and IL2 activated TCRVgamma9(+) lymphocytes and enhanced B-cell depletion from blood and lymph nodes. Thus, the combination with BrHPP PAg is able to improve the efficacy of cancer immunotherapy by therapeutic mAbs.

Antitumor immunity suppresses tumorigenesis, but we do not understand how transformed cells initiate those immune responses that are essential for effective tumor immunosurveillance. Using the 3-MCA fibrosarcoma model, we identified IL-17D as a tumor-expressed cytokine that recruits natural killer cells, leading to the polarization of M1 macrophages and tumor rejection.

Cathelicidins are evolutionarily conserved anti-microbial peptides that have been identified in several epithelial tissues and a wide variety of immune cells. These peptides display multiple host-defense activities, as well as documented but underexplored antitumor activity. Using the regressor MCA sarcoma system as a model of immune mediated tumor rejection, we observed that deficiency in cathelicidin in cnlp-/- mice impaired the rejection of multiple regressor cell lines. Cnlp-/- mice also developed spontaneous lymphoma with age, thus confirming in a separate model system the requirement for cathelicidin in tumor immune surveillance. To decipher the immune defect that may engender impaired tumor surveillance, macrophages from cnlp-/- versus wild type (wt) mice were examined. Interestingly, cnlp-/- macrophages were defective in polarizing towards an antitumor M1 phenotype and less potent at phagocytosing target tumor cells. Altogether, these preliminary data suggest a crucial role of cathelicidin in limiting tumor formation and progression. Future experiments will define the role of cathelicidin in tumor surveillance mediated by macrophages and potentially other innate cells that also express cathelicidin, such as neutrophils and natural killer cells. Citation Format: Emilie T. Gross, Carlos D. Peinado, Isis G. Perez, Samaneh Keshavarz, Jack D. Bui. Cathelicidin is a novel mediator of cancer immune surveillance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1168. doi:10.1158/1538-7445.AM2014-1168

The process of cancer immunoediting generates a repertoire of cancer cells that can persist in immune competent hosts. In its most complex form, this process begins with the elimination of highly immunogenic unedited tumor cells followed by the eventual escape of less immunogenic, edited cells. However, it is not known whether editing can also change the global gene expression profile of developing tumor cells. Here we show using a gene microarray study that the novel cytokine interleukin 17D (IL-17D) is highly expressed in certain unedited but not edited mouse tumor cell lines or certain human tumors. Moreover, forced expression of IL-17D in edited tumor cells induced rejection by stimulating monocyte chemotactic protein 1 (MCP-1, aka CCL2) production from tumor endothelial cells leading to the recruitment of natural killer (NK) cells. We go on to show that NK cells can promote the accumulation of M1-type macrophages. Altogether, these results identify IL-17D as a novel cytokine and describe one mechanism by which IL-17D can induce tumor rejection.

The cancer stem cell (CSC) hypothesis stipulates that tumors follow a hierarchical organization where a subpopulation of cells endowed with stem cell characteristics initiates and maintains the malignant progression. Using the murine MCA sarcoma model system, a widely recognized model for studying cancer immunosurveillance and rejection mechanisms, we sought to explore how hierarchical tumor progression occurs during cancer immunosurveillance. The first step was to define CSC in the MCA sarcoma model. We identified an interesting heterogeneity of the tumor population based on Sca1 and CD90 expression. Purification of the Sca1+ and Sca1+/CD90+ in various cell lines and subsequent in vitro culture demonstrated that the Sca1+/CD90-population is able to regenerate the initial tumor heterogeneity. Transplantation of those two populations in RAGxγc-/- hosts demonstrated either a more rapid onset of sarcoma in mice injected with Sca1+ cells. Interestingly, phenotypic analysis of escaping tumors revealed an increase in Sca1+ cells. Finally, in vitro treatment with IFNg led to a substantial increase in Sca1 expression that correlated with an increase in cells' stemness properties as suggested by the elevated sphere forming capacity of IFNg treated cells. This data suggest that cancer stemness may be modulated by anti-cancer immune responses and may represent an additional mechanism of tumor immune escape.

Our laboratory has recently identified a novel role for the cytokine IL-17D as an important mediator of the antitumor immune response, and the loss of IL-17D as a key component of tumor progression. Our data show for the first time that overexpression of the cytokine IL-17D is sufficient to induce tumor rejection or growth delay. Tumors expressing IL-17D displayed an increased infiltration of natural killer cells, which gave way to a commensurate increase of M1-type macrophages. While examining the regulation of IL-17D, we came across the surprising finding that IL-17D can be induced by oxidative stress signals via the transcription factor nrf2. Nrf2, responsible for regulating the expression of the anti-oxidant response element genes, is known as the primary cellular responder to oxidative stress. Although the role of nrf2 in cancer biology has been well studied, the role of nrf2 in the regulation of immune responses (especially via IL-17D) has yet to be defined. We find that nrf2 is necessary to induce IL-17D expression in a variety of tumor and non-tumor cells, and that this regulation is active in both tumor progression and viral infection. These studies constitute an intriguing and novel connection between oxidative cellular stress and immune activation.