The Role of Autophagy in Salivary Gland Dysfunction Following Targeted Head and Neck Radiation

Head and neck cancer is one of the most common cancers worldwide. The current standard of care for head and neck cancer includes surgical resection of the tumor followed by chemoradiation. This targeted head and neck radiation causes dysfunction of the salivary glands, which leads to xerostomia, mucositis, dysphagia, dental caries, and malnutrition. These side effects greatly decrease patient quality of life and increase their financial responsibility. Current therapies available to ameliorate these negative side effects are expensive, only provide short-term relief, and many of them have negative side effects of their own. Therefore, another therapy is needed to prevent salivary gland dysfunction or restore its function following targeted head and neck radiation. Autophagy is a homeostatic cellular mechanism that could be targeted as a therapeutic mechanism in the salivary glands following targeted head and neck radiation. Autophagy is a catabolic process necessary to maintain cellular homeostasis. It has been shown to play a beneficial role in a variety of disease states including diabetes mellitus, obesity, and cancer. The role of autophagy in the response of cancerous tissue to radiation has been vastly studied. However, the role autophagy plays in normal tissue response to radiation remains poorly understood and much more research in this area is needed.Atg5^(f/f);Aqp5-Cre mice have a conditional knockout of Atg5, a gene necessary for autophagy, in the salivary glands. These mice have unchanged baseline levels of apoptosis, proliferation, and stimulated salivary flow rates when compared to wild-type mice. Therefore, they are a useful model to investigate the role of autophagy in the response of the salivary glands to targeted head and neck radiation. These Atg5^(f/f);Aqp5-Cre autophagy-deficient mice display increased radiosensitivity following targeted head and neck radiation. Furthermore, post-therapy use of CCI-779, a rapalogue and inducer of autophagy, allowed for restoration of salivary gland function following targeted head and neck radiation. Taken together, these results implicate autophagy as playing a beneficial role in normal salivary function following radiation. Therefore, autophagy could be utilized by normal salivary gland tissue following targeted head and neck radiation to maintain salivary gland function

The Rapalogue, CCI-779, Improves Salivary Gland Function following Radiation

<div><p>The standard of care for head and neck cancer typically includes surgical resection of the tumor followed by targeted head and neck radiation. However depending on tumor location and stage, some cases may not require surgical resection while others may be treated with chemoradiation. Unfortunately, these radiation treatments cause chronic negative side effects for patients. These side effects are associated with damage to surrounding normal salivary gland tissue and include xerostomia, changes in taste and malnutrition. The underlying mechanisms of chronic radiation-induced salivary gland dysfunction are unknown, however, in rodent models persistently elevated proliferation is correlated with reduced stimulated salivary flow. The rapalogue, CCI-779, has been used in other cell systems to induce autophagy and reduce proliferation, therefore the aim of this study was to determine if CCI-779 could be utilized to ameliorate chronic radiation-induced salivary gland dysfunction. Four to six week old <i>Atg5^f/f; Aqp5-Cre</i>, <i>Atg5^+/+; Aqp5-Cre</i> and FVB mice were treated with targeted head and neck radiation. FVB mice were treated with CCI-779, chloroquine, or DMSO post-radiation. Stimulated salivary flow rates were determined and parotid and submandibular salivary gland tissues were collected for analyses. Mice with a defect in autophagy, via a conditional knockout of <i>Atg5</i> in the salivary glands, display increased compensatory proliferation in the acinar cell compartment and hypertrophy at 24-72 hours following radiation. FVB mice treated with post-therapy CCI-779 have significant improvements in salivary gland physiology as determined by stimulated salivary flow rates, proliferation indices and amylase production and secretion. Consequently, post-radiation use of CCI-779 allows for improvement of salivary gland function and reestablishment of glandular homeostasis. As CCI-779 is already FDA approved for other uses, it could have a secondary use to alleviate the chronic side effects in head and neck cancer patients who have completed anti-tumor therapy.</p></div

Radiation plus post-therapy CCI-779 improves parotid acinar cell proliferation indices and amylase production levels similar to unirradiated mice.

<p>The head and neck region of FVB mice was exposed to a single 5-Gy dose of radiation and mice received injections of vehicle or CCI-779 on days 4-8 following initial radiation treatment. Parotid salivary glands were then collected 30 days following treatment. Significant differences (<i>p</i><0.05) were determined using an ANOVA followed by a post-hoc Bonferroni multiple-comparison test. Letters above treatment groups are used to signify statistical significance; treatment groups with the same letters are not significantly different from each other. Data are presented as the mean ±SEM. UT: unirradiated. <b>A.</b>) Serial sections were stained for PCNA, a marker of proliferation, and the graph represents the number of acinar cells with positive PCNA staining in the parotid glands as a percentage of the total number of acinar cells. n = 4 per genotype/per treatment. <b>B.</b>) Representative images of positive PCNA staining. <b>C.</b>) Serial sections were stained to determine positive amylase area of the parotid glands. The graph represents the positive amylase area as a percentage of the parotid area as a whole. n = 4 per genotype/per treatment. <b>D.</b>) Stimulated saliva was collected from mice 30 days after treatment with a single 5Gy dose of targeted head and neck radiation and analyzed for total protein content as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113183#s2" target="_blank">Materials and Methods</a>. The graph represents the percentage of amylase protein (ranging from 50–57 kD); n = 10 per genotype/per treatment.</p

Histological changes in parotid and submandibular salivary glands structure following radiation and CCI-779 administration.

<p>The head and neck region of FVB mice was exposed to a single 5-Gy dose of radiation and mice received injections of DMSO vehicle or CCI-779 on days 4-8 following initial radiation. UT: unirradiated. <b>A.</b>) Representative H&E sections were analyzed for differences in salivary gland structure 30 days following radiotherapy. <b>B.</b>) Representative H&E sections were analyzed for differences in salivary gland structure 90 days following radiotherapy.</p

<i>Atg5^f/f;Aqp5-Cre</i> autophagy-deficient mice display increased hyperplasia and elevated compensatory proliferation of the parotid acinar cell population following targeted head and neck irradiation.

<p><i>Atg5^+/+;Aqp5-Cre</i> and <i>Atg5^f/f;Aqp5-Cre</i> (autophagy-deficient) mice were treated with a single 5-Gy dose of targeted head and neck radiation on day 0. At 24, 48, 72 hours and day 30 following treatment, salivary tissue was collected and serial sections were stained. Letters above treatment groups are used to signify statistical significance; treatment groups with the same letters are not significantly different from each other. Significant differences (<i>p</i><0.05) were determined using an ANOVA followed by a post-hoc Bonferroni multiple-comparison test. Data are presented as the mean ±SEM. <b>A.</b>) Representative image of H&E staining of unirradiated <i>Atg5^+/+;Aqp5-Cre</i> control-mice. <b>B.</b>) Representative image of H&E staining of <i>Atg5^f/f</i>;<i>Aqp5-Cre</i> unirradiated mice. <b>C.</b>) Representative image of H&E staining of irradiated <i>Atg5^+/+;Aqp5-Cre</i> mice 30 days after radiotherapy. <b>D.</b>) Representative image of H&E staining of irradiated <i>Atg5^f/f</i>;<i>Aqp5-Cre</i> mice 30 days after radiotherapy. <b>E.</b>) Total acinar cell counts taken from 3–5 images per mouse 30 days after radiotherapy. <i>p</i><0.05; n≥4 per genotype. UT: unirradiated. <b>F.</b>) Serial sections were stained for PCNA, a marker of proliferation, and the graph represents the number of acinar cells with positive PCNA staining in the parotid glands as a percentage of the total number of acinar cells. <i>p</i><0.05, n = 4 per genotype/per treatment. <b>G.</b>) Representative images of positive PCNA staining in the parotid glands.</p

Radiation induces hyperactivation of the mTOR pathway 4–5 days following treatment.

<p>On day 0 FVB wild-type mice were either unirradiated or treated with a single 5-Gy dose of targeted head and neck radiation. Letters above treatment groups are used to signify statistical significance; treatment groups with the same letters are not significantly different from each other. Significant differences (<i>p</i><0.05) were determined using an ANOVA followed by a post-hoc Bonferroni multiple-comparison test. Data are presented as the mean ±SEM. <b>A</b>.) Parotid glands were collected at respective time points and protein was isolated to be used in immunoblots (representative blot shown) and probed for pS706K (top panel), pAkt (middle panel) or total ERK (bottom panel). <b>B</b>.) The graph represents the ratio of phosphorylated S6K to total ERK, which was quantified using densitometry. <i>p</i><0.05, n≥3 per time point/treatment. UT: unirradiated. <b>C</b>.) The graph represents the ratio of phosphorylated Akt to total ERK, which was quantified using densitometry. <i>p</i><0.05, n≥3 per time point/treatment. UT: unirradiated.</p