Purpose Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and FA gene mutations and transcriptional repression are common. Invasive tumor behaviour 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, in order 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 classical hallmarks of FA following exposure to DNA crosslinkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of 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-dependent protein kinase (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.

Purpose Fanconi anemia (FA) is an inherited disorder associated with a constitutional defect in the FA DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with FA are predisposed to formation of head and neck squamous cell carcinomas (HNSCCs) 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 FA patients, and murine HNSCC cell lines derived from the tumors of wild type and Fancc−/− mice, we sought to define FA-dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of FA HNSCC cells for non-homologous end joining (NHEJ). Results Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily FA-dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in FA cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by poly(ADP-ribose) polymerase (PARP) in FA-deficient cells. Moreover, human and murine FA HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by FA gene complementation. Conclusions The observed reliance upon PARP-mediated mechanisms reveals a means by which FA 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.

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.

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.

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.

<p>Figure S3. Effect of DNA-PKcs inhibition on cisplatin sensitivity of HNSCC cells</p>

<p>Figure S4. PARP inhibitor sensitivity of human and murine HNSCC cells</p>

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