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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.

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.

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.

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