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Chick Chorioallantoic Membrane (CAM) assay for UM-SCC1 cells.

Figure S2. Crosslinker and chemotherapy sensitivity of human FA-deficient and -proficient HNSCC lines

Supplementary Materials and Methods. Details on experimental approaches and reagents that were utilized

Cell attachment and migration assays for UM-SCC1 cells.

DNA-PK and Rac1 activity promote UM-SCC47 cell invasion.

RNA sequencing data. Gene ontologies and the associated genes based on biological processes with the most significance based on p-values and corrected q-values.

Purpose: Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells.Experimental Design: Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classic hallmarks of FA following exposure to DNA cross-linkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-dependent protein kinase (DNA-PK) and Rac1 inhibitors.Results: We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-PK and downstream Rac1 GTPase activity.Conclusions: These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes. Clin Cancer Res; 22(8); 2062–73. ©2015 AACR.

Generation and characterization of FA-deficient (HPV positive and HPV negative) HNSCC cell models.

Purpose: Fanconi anemia is an inherited disorder associated with a constitutional defect in the Fanconi anemia DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with Fanconi anemia are predisposed to formation of head and neck squamous cell carcinomas (HNSCC) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease.Experimental Design: Using HNSCC cell lines derived from the tumors of patients with Fanconi anemia, and murine HNSCC cell lines derived from the tumors of wild-type and Fancc−/− mice, we sought to define Fanconi anemia–dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of Fanconi anemia HNSCC cells for non-homologous end joining (NHEJ).Results: Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily Fanconi anemia–dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in Fanconi anemia cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by PARP in Fanconi anemia–deficient cells. Moreover, human and murine Fanconi anemia HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by Fanconi anemia gene complementation.Conclusions: The observed reliance upon PARP-mediated mechanisms reveals a means by which Fanconi anemia HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual. Clin Cancer Res; 21(8); 1962–72. ©2015 AACR.

Legends

Figure S1. Squamous cell carcinoma formation and survival in Fancc-/- and wild type mice

Legends for supplementary figures and table

Table S1. Analyses and crosslinker sensitivity of murine Fancc and wild type cell lines

Figure S4. PARP inhibitor sensitivity of human and murine HNSCC cells

Obesity is becoming a global epidemic and is a risk factor for breast cancer. Environmental enrichment (EE), a model recapitulating an active lifestyle, leads to leanness, resistance to diet-induced obesity (DIO) and cancer. One mechanism is the activation of the hypothalamic–sympathoneural–adipocyte (HSA) axis. This results in the release of norepinephrine onto adipose tissue inducing a drop of leptin. This study aimed to test the effects of EE on breast cancer onset and progression while considering the effect of leptin by utilizing the transgenic MMTV-PyMT model as well as several models of varied leptin signaling. EE was highly effective at reducing weight gain, regardless of the presence of leptin. However, the effects of EE on tumor progression were dependent on leptin signaling. EE decreased leptin and reduced mammary tumor growth rate in MMTV-PyMT spontaneous and DIO transplantation models; in contrast, the absence of leptin in ob/ob mice resulted in increased tumor growth likely due to elevated norepinephrine levels. Our results suggest that the microenvironment is critical in breast tumorigenesis and that the drop in leptin is an important peripheral mediator of the EE anti-breast cancer effects, offsetting the potential pro-tumorigenic effects of norepinephrine responding to a complex environment.

Environmental enrichment (EE), a housing condition providing complex physical, social, and cognitive stimulation, leads to improved metabolic health and resistance to diet-induced obesity and cancer. One underlying mechanism is the activation of the hypothalamic-sympathoneural-adipocyte axis with hypothalamic brain-derived neurotrophic factor (BDNF) as the key mediator. VGF, a peptide precursor particularly abundant in the hypothalamus, was up-regulated by EE. Overexpressing BDNF or acute injection of BDNF protein to the hypothalamus up-regulated VGF, whereas suppressing BDNF signaling down-regulated VGF expression. Moreover, hypothalamic VGF expression was regulated by leptin, melanocortin receptor agonist, and food deprivation mostly paralleled to BDNF expression. Recombinant adeno-associated virus-mediated gene transfer of Cre recombinase to floxed VGF mice specifically decreased VGF expression in the hypothalamus. In contrast to the lean and hypermetabolic phenotype of homozygous germline VGF knockout mice, specific knockdown of hypothalamic VGF in male adult mice led to increased adiposity, decreased core body temperature, reduced energy expenditure, and impaired glucose tolerance, as well as disturbance of molecular features of brown and white adipose tissues without effects on food intake. However, VGF knockdown failed to block the EE-induced BDNF up-regulation or decrease of adiposity indicating a minor role of VGF in the hypothalamic-sympathoneural-adipocyte axis. Taken together, our results suggest hypothalamic VGF responds to environmental demands and plays an important role in energy balance and glycemic control likely acting in the melanocortin pathway downstream of BDNF. (Endocrinology 157: 983-996, 2016)

Purpose: Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells. Experimental Design: Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classic hallmarks of FA following exposure to DNA cross-linkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-dependent protein kinase (DNA-PK) and Rac1 inhibitors. Results: We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-PK and downstream Rac1 GTPase activity. Conclusions: These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes. Clin Cancer Res; 22(8); 2062–73. ©2015 AACR.

Metabolic syndromes instigate substantial morbidity and mortality worldwide via its negative effects of many diseases including cancer. Obesity in particular has been deemed a causative factor for breast cancer in post-menopausal women, while worsening prognosis regardless of menopausal status. Importantly, progression of many metabolic syndromes can largely be slowed or reversed with aggressive lifestyle changes, thus also having the potential to mitigate cancer onset. A mouse model of environmental enrichment (EE) to improve motosensory, cognitive, and social stimulation by increasing physical engagement and social interaction triggers vast improvements in overall health. These include boosting mental health, reducing adiposity, prevention of diet-induced obesity (DIO), promoting the white to brown fat transition, enhancing insulin sensitivity, improving immune function, limiting inflammation, and inhibition of cancer growth. We have elucidated the central mechanism of EE to be the activation of the hypothalamic-sympathoneural-adipocyte (HSA) axis, a specific neuroendocrine route in which the brain communicates with adipose tissue via the sympathetic nervous system. Once activated, norepinephrine (NE) is released directly onto adipose tissue which induces the observed metabolic improvements. Breast cancer is uniquely exposed to this adipose-NE so we sought to explore this distinctive microenvironment in the context of both local and systemic endocrine and immune signaling in varied body mass states and menopausal statuses. Our data showed that EE diminished leptin levels and delayed cancer onset in the MMTV-PyMT spontaneous mouse model of breast cancer and slowed growth in a DIO allograft model. In contrast, EE failed to attenuate tumor progression in ob/ob mice in the absence of leptin, but rather enhanced tumor growth, suggesting that the leptin-NE relationship is one of the key peripheral mediators of the EE anti-breast cancer effects. We also observed significant immune cell modulation in the adipose tissue following EE. Initial characterization revealed a decrease in the proportion of tumor-associated macrophages (TAMs) and an overall anti-inflammatory response which together with the drop in leptin may contribute to the observed anti-cancer effects. We plan to further elucidate this EE-induced adipose and immune cell modulation with the ultimate goal of developing therapeutic strategies to treat both metabolic syndromes as well as associated and possibly independent cancer types. Citation Format: Grant D. Foglesong, Stephen M. Bergin, Wei Huang, Michael A. Caligiuri, Lei Cao. Lifestyle improvements delay cancer onset and progression by adapting endocrine and immune signaling in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1258. doi:10.1158/1538-7445.AM2017-1258

Purpose: Fanconi anemia is an inherited disorder associated with a constitutional defect in the Fanconi anemia DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with Fanconi anemia are predisposed to formation of head and neck squamous cell carcinomas (HNSCC) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease. Experimental Design: Using HNSCC cell lines derived from the tumors of patients with Fanconi anemia, and murine HNSCC cell lines derived from the tumors of wild-type and Fancc−/− mice, we sought to define Fanconi anemia–dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of Fanconi anemia HNSCC cells for non-homologous end joining (NHEJ). Results: Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily Fanconi anemia–dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in Fanconi anemia cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by PARP in Fanconi anemia–deficient cells. Moreover, human and murine Fanconi anemia HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by Fanconi anemia gene complementation. Conclusions: The observed reliance upon PARP-mediated mechanisms reveals a means by which Fanconi anemia HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual. Clin Cancer Res; 21(8); 1962–72. ©2015 AACR.

Obesity affects 35% of U.S. adults and is a leading risk factor for breast cancer in postmenopausal women, but worsens prognosis regardless of menopausal status. Importantly, progression to obesity can largely be slowed or reversed with aggressive lifestyle changes, thus also having the potential to mitigate cancer onset. A mouse model of environmental enrichment (EE) to improve motosensory, cognitive, and social stimulation by increasing physical engagement and social interaction triggers vast improvements in overall health. These include reducing adiposity, promoting the white to brown fat transition, mitigating diet-induced obesity (DIO), and decreasing progression of multiple cancer types including colon cancer and melanoma. We have elucidated the primary mechanism of the EE-induced phenotype to be the activation of the hypothalamic-sympathoneural-adipocyte (HSA) axis, a specific neuroendocrine route in which the brain communicates with adipose tissue. These benefits may also be partially attributable to the sharp drop in serum leptin levels following EE which has been implicated in diminishing tumorigenesis, invasiveness, and metastasis. Here we investigated the effects of EE on breast tumorigenesis in all body mass states and menopause-associated hormone conditions. Our preliminary studies showed that EE delayed the cancer onset in the MMTV-PyMT spontaneous mouse model of breast cancer. In addition the role of leptin signaling in the EE-induced effects on breast tumorigenesis was investigated by utilizing obese models with varied leptin signaling including leptin receptor-defective db/db mice that express extremely high levels of leptin, leptin-defective ob/ob mice that do not express leptin but are leptin-sensitive, and a diet-induced obesity (DIO) model with elevated leptin but intact leptin signaling. These obese mice were housed in EE or control housing until significantly attenuated weight gain was observed followed by implantation of primary PyMT-derived mammary tumor cells. EE was highly effective at reducing adiposity in each of these obesity models, regardless of leptin signaling. However, the effects of EE on mammary tumor progression were dependent on leptin signaling. EE decreased leptin level and inhibited mammary tumor growth in DIO mice. In contrast EE failed to attenuate tumor progression in ob/ob mice in the absence of leptin, suggesting that leptin is a key mediator of the EE anti-cancer effects on breast cancer. The elucidation of mechanisms of EE and the HSA axis-induced improvement in overall health will provide effective prevention as well as therapeutic strategies for metabolic syndromes and associated cancer types. Citation Format: Grant Foglesong, Wei Haung, Lei Cao. Leptin mediates the anti-breast cancer effects of environmental enrichment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4176.