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. Author manuscript; available in PMC: 2023 Apr 1.
Published in final edited form as: Exp Mol Pathol. 2022 Feb 18;125:104752. doi: 10.1016/j.yexmp.2022.104752

PI3K/mTOR Inhibition Prevents Anal Cancer in Mice with Established Low-grade Anal Dysplasia

Laura C Gunder a, Tyra H Moyer b, Brooks L Rademacher a, Andrew S Auyueng c, Glen Leverson a, Wei Zhang d,e, Kristina A Matkowskyj d,e,f, Evie H Carchman a,e,f,*
PMCID: PMC9018484  NIHMSID: NIHMS1782942  PMID: 35183509

Abstract

Low-grade anal dysplasia is a disease that can progress to high-grade anal dysplasia and eventually anal cancer if left untreated. Research has shown that low-grade anal dysplasia is marked by significant autophagic dysfunction. We hypothesized that systemic induction of autophagy, via phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibition, would be effective in preventing anal cancer development in human papillomavirus (HPV) mice (K14E6/E7) with established low-grade anal dysplasia. Mice began treatment at 15 weeks of age, when 75% of mice spontaneously develop low-grade anal dysplasia, and were divided into the following groups: no treatment, systemic LY3023414 (4.5mg/kg, dual PI3K/mTOR inhibitor) alone, topical 7,12 dimethylbenz[a]anthracene (DMBA) alone, or systemic LY3023414 and topical DMBA. Groups were compared for final histology, PI3K activity, mTOR activity, autophagic induction (light chain 3B (LC3β)), autophagic function (p62 protein), and tumor-free survival. Untreated mice or mice treated with LY3023414 alone did not progress to cancer. There was a statistically significant decrease in the number of mice that developed histologic evidence of cancer when comparing mice that received systemic LY3203414 with topical DMBA versus those that received topical DMBA alone (p=0.0003). PI3K and mTOR activity decreased in groups treated with systemic LY3023414 and topical DMBA as compared with those treated with topical DMBA alone (p= 0.0005 and p= 0.0271, respectively). LC3β and p62 expression was not statistically altered with systemic LY3023414 treatment. Mice developed less overt tumors and had increased tumor-free survival when treated with systemic LY3023414 in the presence of topical DMBA compared to topical DMBA alone (p= 0.0016 and p <0.001, respectively). Systemic LY3023414 treatment is effective in anal cancer prevention in the setting of established low-grade anal dysplasia in an HPV-associated mouse model of anal cancer.

Keywords: anal dysplasia, anal cancer, human papillomavirus, cancer prevention, autophagy, phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR)

1. Introduction

In 2021 alone, an estimated 9,090 patients in the United States were diagnosed with squamous cell carcinoma of the anus (hereafter “anal cancer”), and approximately 1,430 died from the disease (SEER, 2021). For the last two decades, the incidence and mortality of anal cancer in the United States has increased yearly (2.7% and 3.1%, respectively), with the greatest increase occurring in patients infected with the human immunodeficiency virus (HIV) (Deshmukh et al., 2020; Zizza et al., 2021).

The primary risk factor for the development of anal cancer is persistent infection with a high-risk strain of human papillomavirus (e.g., HPV16, HPV18), which is prevalent in HIV-positive patients (Lin et al., 2018; Thompson et al., 2018). HPV infection causes transformation of normal anal epithelium to low-grade anal squamous dysplasia, which then progresses to high-grade anal squamous dysplasia, and ultimately to invasive anal squamous cell carcinoma. Dysplasia refers to abnormal cellular growth with an increased likelihood for progression to carcinoma. Current treatments for anal dysplasia include localized destruction of anal lesions or non-specific stimulation of the immune system to clear HPV. These treatments are labor intensive, non-specific (affecting both diseased and non-diseased tissue), and cause significant side effects (e.g.,anal pain, anal stenosis) (Leeds and Fang, 2016).

There are no established national guidelines for treatment of anal dysplasia, primarily due to a lack of effective or well-tolerated therapies. Topical application of 5-fluorouracil (5-FU) or Imiquimod, current topical treatment options for anal dysplasia, kills cells indiscriminately and/or induces inflammation in both normal and diseased tissues. The topical treatments often result in significant discomfort for patients, thus leading to poor patient compliance. Curative rates using these therapies are low, with complete response rates of only 17% (5-FU) and 24% (Imiquimod). Furthermore, 27% of 5-FU users and 43% of Imiquimod users experience grade 3–4 toxicities (Elorza et al., 2016). Destructive techniques (ablation/excision) of precancerous lesions are comparable to those performed for cervical dysplasia and are invasive, painful, and can result in long-term anorectal dysfunction (e.g., fecal incontinence and anal stenosis). Finally, all these treatment options have high recurrence rates (>50%) and require frequent costly surveillance (Leeds and Fang, 2016). Thus, there is a significant need for effective,targeted therapies with a low side-effect profile.

Promising results have been found through the use of dual phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitors, which show significant reductions in anal carcinogenesis through the activation of autophagy in an HPV mouse model with starting normal histology (Rademacher et al., 2017; Rademacher et al., 2019). It has been previously demonstrated that autophagic dysfunction occurs early on in anal carcinogenesis in the presence of low-grade anal dysplasia, with normalization in the later stages (high-grade anal dysplasia and anal cancer) (Carchman et al., 2016). Autophagy is a well-conserved, intracellular, catabolic process where damaged and/or unused organelles and proteins are isolated in a double-membrane vesicle called an autophagosome (Klionsky et al., 2016; Levine and Klionsky, 2004; Wang and Klionsky, 2003). The autophagosome is marked by the autophagic protein light chain 3B (LC3β) and then fuses with the lysosome to allow for degradation of its contents, including the ubiquitin-binding protein (p62), which is degraded solely via the autophagic pathway (Moscat and Diaz-Meco, 2009). There are several processes and pathways that regulate autophagic induction and function to tightly control the metabolic homeostasis within each eukaryotic cell (Lum et al., 2005). The PI3K and mTOR pathways are the main upstream effectors of autophagy and have been shown to be relevant targets for therapeutic intervention (Chen and Karantza-Wadsworth, 2009; Kondo et al., 2005; Martelli et al., 2012; Shin et al., 2019).

In this study, we examine the role of a pathway-inhibiting agent, LY3023414, which targets both the PI3K and mTOR pathways in order to prevent anal cancer development in an HPV mouse model with animals that have established low-grade anal dysplasia. We hypothesized that targeting autophagy in the setting of low-grade anal dysplasia would prevent anal cancer development and have a minimal side-effect profile

2. Materials and Methods

2.1. K14E6/E7 Mice

K14E6/E7 mice were generated as previously described in Stelzer et al. (2010). These mice express the HPV16 oncoproteins, E6 and E7, in their epithelium. The mice spontaneously develop anal dysplasia which can progress into invasive squamous cell carcinoma, similar to that seen in humans. This progression is accelerated when topically treated with the carcinogen, 7, 12 dimethylbenz[a]anthracene (DMBA). A time-course study from previous work determined that at 15 weeks of age without DMBA treatment, greater than 75% of the transgenic mice develop low-grade anal dysplasia (Carchman et al., 2016). All mice were maintained in an American Association for Accreditation of Laboratory Animal Care-approved Wisconsin Institute for Medical Research (WIMR) Animal Care Facility. Experiments were performed in accordance with approved Institutional Animal Care and Use Committee protocol M005946.

2.2. Treatment with 7, 12 dimethylbenz[a]anthracene (DMBA)

Treatment with DMBA has been shown to synergize with HPV oncogenes to induce tumorigenesis (Song et al., 2000). Weekly topical application of 0.12 μmole DMBA (60% acetone/40% dimethyl sulfoxide (DMSO)) to the anus of K14E6/E7 mice was performed (Carchman et al., 2016; Stelzer et al., 2010) starting at 15 weeks of age. Mice were monitored weekly for tumor development and growth. Animals were sacrificed at 20 weeks post initiation of DMBA treatment (35 weeks of age). This time point was chosen because with 20 weeks of DMBA treatment, 100% of animals have been shown to have overt anal cancer (Carchman et al., 2016). Control mice were age-matched K14E6/E7 mice not treated with topical DMBA or systemic LY3023414.

2.3. Treatment with systemic LY3023414

LY3023414 (Medchem Express, NJ, USA) was given to 25 K14E6/E7 mice (4.5 mg/kg in 1% hydroxyethyl cellulose) (Smith et al., 2016) was given to K14E6/E7 mice via oral gavage, five days per week (Monday-Friday) for 20 weeks, starting at 15 weeks of age ( Figure 1). Approximately half of these mice were treated with topical DMBA to the anus once weekly for 20 weeks, in conjunction with systemic LY3023414, while the other half were not treated with topical DMBA.

Figure 1.

Figure 1.

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K14E6/E7 mice anal tissue after 2-week treatment with varying concentrations of LY3023414, to determine lowest dosage that results in PI3K and mTOR inhibition. The sections of tissue are immunohistochemically stained for pAKT (A-E) or pS6 (F-J). Tissue was reviewed under light microscope and 200x magnification images were acquired using the Zeiss Axio Imager M2 imaging system. These images demonstrate that 1% LY3023414 was the lowest concentration that provided the greatest inhibition of PI3K and mTOR based on decreased levels of pAKT and pS6.

2.4. Mouse Observations

Mice underwent daily observation to determine local side effects from both systemic LY3023414 and topical DMBA treatment (hair loss, skin irritation, diarrhea, etc.). Mice were checked weekly for overt anal tumor development until sacrifice.

2.5. Histology

Anal tissue was collected and fixed in 4% paraformaldehyde for 24 hours, then placed in 70% ethanol. After fixation, the tissues were processed, embedded in paraffin, and serially sectioned at 5 μm thickness. Sections were stained with hematoxylin and eosin (H&E) and evaluated by a gastrointestinal pathologist for evidence of anal dysplasia (low-grade versus high-grade) or invasive carcinoma.

2.6. Immunohistochemistry (IHC)

The protein expression of phosphorylated-Serine473-AKT (pAKT) and phosphorylated S6 (pS6) were assessed using immunohistochemistry, as they are downstream products of PI3K and mTOR activation, respectively. Paraffin-embedded sections were deparaffinized, rehydrated, subjected to citrate buffer antigen retrieval, permeabilized with 2N HCl, and then blocked with 5% horse serum. Sections were then stained with a monoclonal rabbit antibody against pAKT (1:50 in 5% horse serum in phosphate buffered saline (PBS); antibody #3787, Cell Signaling Technology) or a polyclonal rabbit antibody against pS6 (1:50 in 5% horse serum in PBS; antibody #2211, Cell Signaling Technology) overnight at 4° Celsius. Each slide was then washed and incubated with VectaStain universal secondary antibody (1:50 in 5% horse serum in PBS; Vector Laboratories). Slides were then subjected to R.T.U. VectaStain ABC reagent (Vector Laboratories, Inc. Bulingame, CA). Finally, sections were incubated with 3, 3’-diaminobenzidine (DAB) substrate (DAB Peroxidase (HRP) Substrate; Vector Laboratories, Inc. Bulingame, CA); counterstained with Vector Hematoxylin QS (H-3404, Vector Laboratories, Inc. Bulingame, CA); dehydrated; and cover slipped. Light microscopy was performed and images were acquired using a 20x objective on a Zeiss Axio Imager M2 imaging system. Images were then analyzed with ImageJ.

2.7. Immunofluorescence (IF)

Anal tissue sections were deparaffinized, rehydrated, subjected to antigen retrieval, permeabilized, and blocked with 10% donkey serum per standard protocol (Barth et al., 2010). To examine autophagy, the sections were stained with monoclonal rabbit antibody against LC3β (1:50 in 10% donkey serum in PBS; Santa Cruz Biotechnology) or monoclonal mouse antibody for p62 (1:200 in 10% donkey serum in PBS; Abcam) overnight at 4° Celsius. Sections were then washed and stained with donkey anti-rabbit Fluor 488 and donkey anti-mouse Fluor 594 (1:500 in 10% donkey serum in PBS; Life Technologies) for one hour in the dark at room temperature. Slides were then counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Slides were imaged using the Zeiss Axio Imager M2 imaging system. Images at 20x objective were obtained. Each 200x image was imported into Image J version 2.0.0 (Fiji distribution) and underwent the following processing: Images were split into the three channels (488, 594, and DAPI). All images were thresholded using the default dark background. The area of interest, the anorectal transition zone (ATZ), was manually selected and then RawIntDen (raw integrated density) values were measured for the region of interest to measure the intensity of the fluorescent signal. The RawIntDen was then normalized for the area of the region selected (RawIntDen/Area).

2.8. Statistical analysis

In order to detect at least a 50% difference in tumor incidence with a Type I error rate of 5% and a Type II error rate of 20% (80% power) between the treated and untreated groups, 12 mice per group were needed. Chi-Square Test or Fisher’s two-sided exact tests were used to establish an association between treatment groups and histological grade of disease/outcome at 20 weeks. The comparisons between treatment groups in terms of expression of pAKT, pS6, LC3β, and p62, were made using Fisher’s protected least significant differences (LSD) tests. Fisher’s exact tests were used to determine relationships between treatment groups with their respective controls and final outcome of overt anal tumor development. A Kaplan-Meier analysis with Log-Rank testing was applied to determine differences in time to overt anal tumor onset. If comparison tests failed the test of equal variance, tests were rerun based on log expressions. SAS versus 9.4 (SAS Institute, Inc.,Cary, NC) was utilized to perform each of these analyses. Statistical significance was defined as p-value ≤ 0.05.

3. Results

3.1. LY3023414 treatment did not result in local side effects

In terms of side effects from systemic therapy with LY3023414, there was no evidence of local or systemic adverse reactions such as hair loss, erythema, irritation, or diarrhea.

3.2. Treatment with systemic LY3023414 reduced rates of anal cancer on histology

There was a significant association between all treatment groups and histological outcome (p<0.0001). There was not a significant difference between control and systemic LY3023414 alone groups in terms of histological outcome (cancer on histology). After the 20-week treatment period, 33.3% (10/30) of the control mice had low-grade dysplasia of the anus on final histology while 50% (15/30) progressed to high-grade anal dysplasia and 16.6% (5/30) regressed to normal histology. In those treated with systemic LY3023414 alone, 16.7% (2/12) were noted to have low-grade anal dysplasia, 66.7% (8/12) had high-grade anal dysplasia, and 16.7% (2/12) had normal histology. There was a significant decrease in the histological grade of disease in mice treated with systemic LY3023414 with topical DMBA versus topical DMBA alone (p=0.0003). For the mice that were treated with topical DMBA alone for 20-weeks, 96.8% (31/32) had histologically proven anal cancer, while only one (1/32) mouse had high-grade anal dysplasia. Finally, for the mice treated with both systemic LY3023414 and topical DMBA for 20-weeks, 53.8% (7/13) developed anal cancer and 46.2% (6/13) had high-grade anal dysplasia (Figure 2A). Thus, there was a statistically significant reduction in rates of cancer development with systemic LY3023414 treatment in combination with topical DMBA as compared to DMBA treatment alone (p= 0.0012). Please refer to Figure 2B for representative histology slides (400x) for each treatment group.

Figure 2.

Figure 2.

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Treatment with LY3023414 in mice given DMBA decreased microscopic cancer

Figure 2A. Microscopic Histology of K14E6/E7 mice that started with low-grade anal dysplasia, after 20 weeks of treatment: no treatment, DMBA alone, systemic LY3023414 alone, systemic LY3023414, and DMBA. The notation * represents statistical significance (p <0.05).

Figure 2B. Representative hematoxylin and eosin-stained anal tissues from mice after completing the 20-week treatment period starting with low-grade anal dysplasia. Images taken at 40x magnification. A. received no treatment--low-grade with a focus of high-grade, B. received DMBA alone--SqCC, Grade 1, C. received systemic LY3023414 alone--low-grade dysplasia, and D. received systemic LY3023414 and DMBA--high-grade dysplasia.

3.3. Systemic LY3023414 successfully inhibited PI3K and mTOR pathways

Immunohistochemistry for pAKT and pS6 was performed to assess the target effects of systemic LY3023414 on PI3K and mTOR, respectively: pAKT is a known marker for an active PI3K response and pS6, is a known marker for an active mTOR response. With regards to pAKT, there was a significant decrease in protein expression when comparing topical DMBA alone to the systemic LY3023414 and topical DMBA treatment group (mean values= 0.25±0.12 vs 0.10±0.07; p= 0.0005; Figure 3A). There was significant decrease in pAKT expression when comparing the no treatment group to treatment with systemic LY3023414 alone and the systemic LY3023414 and topical DMBA treatment groups (mean values= 0.29±0.15 vs 0.18±0.14 and 0.29±0.15 vs 0.10±0.07; p=0.0150,p<0.0001, respectively; Figure 3A)). These data indicate proper inhibition of the PI3K pathway through use of systemic LY3023414.

Figure 3.

Figure 3.

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Treatment with systemic LY3023414 effectively decreased PI3K and mTOR Activity

Figure 3A. Effect of systemic LY3023414 on pAKT protein expression quantified via immunohistochemical staining of the mouse anorectal transition zone. Mean± standard deviation values are reported in the RawIntDen/Area (raw integrated density/area ) of intensity signal. The notation * represents statistical significance (p <0.05).

Figure 3B. Effect of systemic LY3023414 on pS6 protein expression quantified via immunohistochemical staining of the mouse anorectal transition zone. Mean ± standard deviation values are reported in RawIntDen/Area (raw integrated density/area) of intensity signal.The notation * represents statistical significance (p <0.05).

Figure 3C. Sections of K14E6/E7 mice anal tissue after the 20-week treatment period, immunohistochemically stained for pAKT (A-D) or pS6 (E-H). A,E. no treatment, B,F. topical DMBA alone, C,G. systemic LY3023414 alone and D,H. systemic LY3023414 and topical DMBA.

Comparable to pAKT, there was significant reduction in pS6 expression in systemic LY3023414 alone and systemic LY3023414 and topical DMBA treatment groups as compared to the no treatment group (mean values= 0.13±0.05 vs 0.22±0.10 and 0.17±0.07 vs 0.22±0.10; p=0.0018, p=0.0459, respectively; Figure 3B). There was also a significant decrease in pS6 levels seen in systemic LY3023414 alone as compared to the topical DMBA alone (mean values= 0.13±0.05 vs 0.20±0.08. p= 0.0271;Figure 3B).These data indicate inhibition of the mTOR pathway with systemic LY3023414.

3.4. Treatment with systemic LY3023414 trended towards increased autophagic induction and function

Immunofluorescence for LC3β and p62 was performed to assess autophagic induction and function, respectively. With regard to LC3β, there was only a significant decrease in autophagic induction between topical DMBA alone treatment as compared to the no treatment group(mean values= 3.81±2.09 vs. 6.95±3.63; p =0.0001;Figure 4A). Although not statistically significant, treatment with systemic LY3023414 alone trended towards increased autophagic induction when compared to the no treatment group (mean values= 5.20±2.98, 6.95±3.63; p= 0.1371; Figure 4A) Comparison tests failed the test of equal variance; p-values are based on log expressions.

Figure 4.

Figure 4.

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Treatment with systemic LY3023414 with or without topical DMBA appeared to increase autophagic induction and function as compared to controls

Figure 4A. Effect of systemic LY3023414 on LC3β protein expression quantified via immunofluorescent staining of the mouse anorectal transition zone. Mean ± standard deviation values are reported in RawIntDen/Area (raw integrated density/area) of fluorescent signal. The notation * represents statistical significance (p <0.05).

Figure 4B. Effect of systemic LY3023414 on p62 protein expression quantified via immunofluorescent staining of the mouse anorectal transition zone. Mean ± standard deviation values are reported in RawIntDen/Area (raw integrated density/area) of fluorescent signal. In Figure 4B, for systemic LY3023414 and topical DMBA treatment groups, the median is equal to the first quartile. The notation * represents statistical significance (p <0.05).

Figure 4C. Sections of K14E6/E7 mice anal tissue after the 20-week treatment period, immunofluorescent stained for LC3β (green) and p62 (red).

There was a significant decrease in p62 levels, indicating increased autophagic function, when comparing the no treatment group to both topical DMBA alone and systemic LY3023414 and topical DMBA groups (mean values= 8.58±5.01 vs 4.99±2.66 and 8.58±5.01 vs 5.25±2.79; p=0.0010 and p= 0.0353, respectively; Figure 4B). Comparison tests failed the test of equal variance; p-values are based on log expressions.

3.5. Co-treatment with systemic LY3023414 and topical DMBA results in decreased and delayed anal tumor formation

Mice in the no treatment or systemic LY3023414 alone groups did not develop any overt anal tumors during the 20-week treatment period. There was a statistically significant increase in tumor-free survival when comparing systemic LY3023414 and topical DMBA to the topical DMBA alone group (p= 0.0016). In mice treated with topical DMBA alone, 88.0% (28/32) developed overt anal tumors over the 20-week treatment period and of the 28 mice that developed tumors, the average time to initial tumor appearance was 15.25± 3.16 weeks into treatment. Only 23.1% (3/13) of the systemic LY3023414 and topical DMBA-treated mice developed overt anal tumors, and those three mice developed tumors in an average of 17.67±2.08 weeks into the treatment period, with an additional two mice noted to have microscopic foci of invasive cancer on histology. (Figure 5).

Figure 5.

Figure 5.

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The Kaplan Meyer survival analysis indicated prolonged tumor-free survival in mice given systemic LY3023414 with topical DMBA as compared to topical DMBA alone treated mice with starting low-grade anal histology. The green-blue line shows where no treatment and systemic LY3023414 alone groups overlap. The notation * represents statistical significance (p <0.05) between DMBA alone and LY3023414 and DMBA groups (yellow and black lines, respectively).

4. Discussion

In order to prevent anal cancer and to identify novel agents for the treatment of anal dysplasia, a better understanding of the molecular pathways leading to the progression of anal dysplasia to carcinoma is needed. Using an HPV16 E6/E7 transgenic mouse model, we previously demonstrated that the development of anal cancer can be prevented using a dual PI3K/mTOR inhibitor (Rademacher et al., 2019). PI3K and mTOR pathways are active in all cells and function to promote cell survival. Specifically, PI3K functions to stimulate cell growth and proliferation as well as cell cycle progression. mTOR regulates gene transcription and protein synthesis, which in turn regulates cell proliferation (Cantley, 2002, Guertin and Sabatini, 2007, Porta et al., 2014). Dysregulation of the PI3K and mTOR pathways have been implicated in many human cancers (Alzahrani 2019)

This study shows that the systemic use of the dual PI3K/mTOR inhibitor, LY3023414, was effective in inhibiting PI3K and mTOR pathways as assessed by expression of pAKT and pS6, comparable to prior studies with BEZ235 (Rademacher et al. 2019). Systemic LY3023414 was effective in slowing the progression of anal carcinogenesis, as mice that start with low-grade anal dyplasia when given topical DMBA in conjunction with systemic LY3023414 develop significantly less carcinoma compared to carcinogen alone.

Though the dual PI3K/mTOR inhibitor LY3023414 was effective in preventing anal cancer development from established low-grade anal dysplasia, dual PI3K/mTOR inhibitors may not be effective in more advanced stages of dysplasia; thus, the use of dual inhibitors may only be beneficial in patients presenting low grade anal dysplasia or normal histology. As previously stated, autophagic dysfunction occurs early on in the presence of low-grade dysplasia and normalizes as dysplasia progresses to cancer in mice (Carchman et al., 2016). Though autophagy has significant anti-tumor effects, dysregulating autophagy at more advanced stages of disease has been shown to contribute to tumorigenesis. This is corroborated by the fact that autophagy can either be beneficial or deleterious, providing cytoprotection and/or cell recycling (Marinković et al., 2018). It is important to take into consideration the clinical relevance when examining patients with high-grade anal dysplasia and providing recommendations for dual PI3K/mTOR inhibitor therapy.

Unlike our prior studies with BEZ235, this study did not highlight a significant upregulation in autophagy. As described in Rademacher et al. (2019), treatment with topical application of BEZ235 significantly increased LC3β protein expression and reduced p62 expression, representing marked autophagic induction and function. Though treatment with systemic LY3023414 trended towards increased autophagic induction and function, treatment did not significantly alter either marker of autophagy. The difference in autophagic expression between these studies may be due to the variation in the drugs themselves as well as the method of delivery and tissue concentrations. Systemic application via oral gavage will likely provide a less localized treatment response as compared to directly applying the drug to the area of interest. Future studies will focus on the topical application of LY3023414 to the anus in mice with varying established anal histologies: normal histology, low-grade anal dysplasia, and high-grade anal dysplasia.

Given the small size of the anorectal transition zone, this study is limited by not having sufficient tissue to perform western blot analysis to quantify markers of the PI3K/mTOR pathway and autophagy. To address this issue, a power analysis was performed to confirm that the number of mice in each group would allow for proper determination of significance between protein quantification endpoints. The second limitation is that we do not know the systemic and tissue concentrations of the drug in this study.

5. Conclusions

Treatment with a systemic dual PI3K/mTOR inhibitor, LY3023414, can reduce rates of cancer in an HPV-associated transgenic mouse model where the mice have established low-grade anal dysplasia. Systemic LY3023414 delayed the onset of overt anal tumors and reduced overall development of tumors even in the presence of the carcinogen, DMBA. This effect does not appear to be correlated with autophagic induction or function.

Highlights.

  • Treatment with a systemic dual PI3K/mTOR inhibitor, LY3023414, reduced the rate of microscopic anal cancer in HPV transgenic mice with established low-grade anal dysplasia.

  • The delay in progression of low-grade anal dysplasia to cancer using LY3023414 correlated with evidence of PI3K/mTOR pathway inhibition based on downstream targets.

  • The delay in progression of low-grade anal dysplasia to cancer using a dual PI3K/mTOR inhibitor did not correlate with markers of autophagic induction or function.

  • Systemic LY3023414 increased tumor-free survival, even in the presence of the carcinogen, 7, 12 dimethylbenz[a]anthracene (DMBA).

Acknowledgements

We would like to acknowledge Dr. Paul Lambert for his generous support, and for providing the mice utilized in these experiments. The authors thank the University of Wisconsin Carbone Cancer Center (UWCCC) Experimental Pathology Laboratory, a shared research core supported by P30 CA014520, for use of its facilities and services. Thank you to contributing students in the laboratory at the Department of Surgery, Michael T. Bean, Margaret A. Finlay, and Jackie Chang, as well as to Dana Maya and Dr. Hillary Johnson for proofreading.

Funding:

This work is supported by the Society for Surgery of the Alimentary Tract [grant number PRJ96MZ]; the American Society of Colon and Rectal Surgeons [grant number AAC5299]; the National Cancer Institute of the National Institutes of Health [grant number T32CA090217]; and the University of Wisconsin Carbone Cancer Center [grant number P30 CA014520].

Abbreviations:

5-FU

5-fluorouracil

ATZ

anorectal transition zone

DAB

3, 3’-diaminobenzidine

DAPI

4′,6-diamidino-2-phenylindole

DMBA

7,12 dimethylbenz[a]anthracene

DMSO

dimethylsulfoxide

H&E

hematoxylin and eosin

HCl

hydrochloric acid

HIV

human immunodeficiency virus

HPV

Human papillomavirus

LC3β

protein light chain 3 beta

LSD

least significant differences

mTOR

mammalian(mechanistic) target of rapamycin

pAKT

phosphorylated AKT

PBS

phosphate buffered saline

PI3K

phosphoinositide 3 kinase

pS6

phosphorylated S6

RawIntDen

raw integrated density

Footnotes

Declarations of interest: none

CRediT author statement

Laura C. Gunder: Investigation, Resources, Data Curation, Formal Analysis, Writing-Original Draft, Writing- Review & Editing, Supervision, Project Administration. Brooks L. Rademacher: Investigation, Data Curation, Funding Acquisition. Tyra H. Moyer: Investigation, Resources, Data Curation, Writing- Review & Editing Andrew S. Auyeung: Investigation, Resources, Visualization, Supervision. Glen E. Leverson: Formal Analysis, Writing-Original Draft. Wei I. Zhang: Formal Analysis. Kristina A. Matkowskyj: Formal Analysis. Evie H. Carchman: Conceptualization. Methodology, Validation, Formal Analysis, Writing-Original Draft, Writing- Review & Editing, Supervision, Project Administration, Funding Acquisition.

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