Preclinical Models of Anal Cancer Combined-Modality Therapy.

INTRODUCTION: There have been no significant changes in anal cancer treatment options in 4 decades. In this study, we highlight two preclinical models designed to assess anal cancer treatments. MATERIALS AND METHODS: Transgenic K14E6/E7 mice were treated with 7, 12-dimethylbenz(a)anthracene until anal tumors developed. Mice were treated with localized radiation in addition to chemotherapy (combined-modality therapy [CMT]) and compared to no treatment control (NTC). K14E6/E7 mouse anal spheroids with and without Pik3ca mutations were isolated and treated with vehicle, LY3023414 (LY3) (a drug previously shown to be effective in cancer prevention), CMT, or CMT + LY3. RESULTS: In the in vivo model, there was a significant increase in survival in the CMT group compared to the NTC group (P = 0.0392). In the ex vivo model, there was a significant decrease in the mean diameter of CMT and CMT + LY3-treated spheroids compared to vehicle (P ≤ 0.0001). For LY3 alone compared to vehicle, there was a statistically significant decrease in spheroid size in the K14E6/E7 group without mutation (P = 0.0004). CONCLUSIONS: We have provided proof of concept for two preclinical anal cancer treatment models that allow for the future testing of novel therapies for anal cancer.

Topical Protease Inhibitor Decreases Anal Carcinogenesis in a Transgenic Mouse Model of HPV Anal Disease.

Anal cancer is a major health problem. This study seeks to determine if the topical protease inhibitor Saquinavir (SQV), is effective at the prevention of anal cancer in transgenic mice with established anal dysplasia. K14E6/E7 mice were entered into the study when the majority spontaneously developed high-grade anal dysplasia. To ensure carcinoma development, a subset of the mice was treated with a topical carcinogen: 7,12-Dimethylbenz[a]anthracene (DMBA). Treatment groups included: no treatment, DMBA only, and topical SQV with/without DMBA. After 20 weeks of treatment, anal tissue was harvested and evaluated histologically. SQV was quantified in the blood and anal tissue, and tissue samples underwent analysis for E6, E7, p53, and pRb. There was minimal systemic absorption of SQV in the sera despite high tissue concentrations. There were no differences in tumor-free survival between SQV-treated and respective control groups but there was a lower grade of histological disease in the mice treated with SQV compared to those untreated. Changes in E6 and E7 levels with SQV treatment suggest that SQV may function independently of E6 and E7. Topical SQV decreased histological disease progression in HPV transgenic mice with or without DMBA treatment without local side effects or significant systemic absorption.

The Use of the Protease Inhibitor, Saquinavir, to Treat Anal Cancer Spheroids Derived From Human Papillomavirus Transgenic Mice.

BACKGROUND: Anal cancer is associated with high-risk human papillomavirus infection and oncoprotein expression. We have identified several protease inhibitors, used to treat HIV, that decrease oncogene expression. OBJECTIVE: The aim of this project is to determine whether saquinavir, a protease inhibitor, results in a treatment response in anal cancer spheroids. DESIGN: K14E6/E7 transgenic mice (n = 5), which express human papillomavirus 16 oncoproteins E6 and E7 in their epithelium, were treated topically at the anus with a carcinogen, 7,12-dimethylbenz[a]anthracene, to promote anal tumor growth. Tumors were excised and digested, and cells were plated. The tumor cells form 3D multicellular aggregates known as spheroids. SETTINGS: This study was performed in an American Association for Accreditation of Laboratory Animal Care-approved facility. INTERVENTIONS: Spheroids were placed in treatment groups: no treatment, vehicle (dimethyl sulfoxide), and 15 µM saquinavir. Spheroids were imaged immediately pretreatment and 24 hours posttreatment. MAIN OUTCOME MEASURES: Spheroid diameters were measured using ImageJ and mean percent reduction was calculated for each spheroid to determine treatment effect on spheroid growth. Analysis of variance using pairwise comparisons was performed with Fisher protected least significant difference tests. RESULTS: The no-treatment (n = 119 spheroids) and vehicle (n = 126 spheroids) groups demonstrated an increase in spheroid diameter during the treatment period. In contrast, spheroids treated with saquinavir (n = 151 spheroids) demonstrated a statistically significant percent reduction compared to the no-treatment ( p < 0.0001) and vehicle ( p = 0.002) groups. LIMITATIONS: A limitation of these data is that some human error is likely present given that images were analyzed by 3 different scientists. CONCLUSIONS: Saquinavir leads to a statistically significant percent reduction in mice anal tumor spheroid growth ex vivo compared to control groups. Protease inhibitor therapy may be an effective treatment or adjuvant therapy to the Nigro protocol to promote anal cancer tumor regression. See Video Abstract at http://links.lww.com/DCR/C82 . EL USO DEL INHIBIDOR DE LA PROTEASA, SAQUINAVIR, PARA TRATAR LOS ESFEROIDES DEL CNCER ANAL DERIVADOS DE RATONES TRANSGNICOS PARA EL VPH: ANTECEDENTES:El cáncer anal está asociado con la infección por el virus del papiloma humano de alto riesgo y la expresión de oncoproteínas. Hemos identificado varios inhibidores de la proteasa, utilizados para tratar el VIH, que disminuyen la expresión del oncogén.OBJETIVO:El objetivo de este proyecto es determinar si los esferoides de cáncer anal responden al tratamiento con inhibidor de la proteasa, Saquinavir.DISEÑO:Ratones transgénicos K14E6/E7 (n = 5), que expresan las oncoproteínas E6 y E7 del VPH16 en su epitelio, fueron tratados tópicamente en el ano con carcinógeno, 7,12 dimetilbenz[a]antraceno, para promover el crecimiento del tumor anal. Los tumores se extirparon y digirieron, y las células se sembraron en placas. Las células tumorales forman agregados multicelulares tridimensionales, conocidos como esferoides.ESCENARIO:Este estudio se realizó en un centro aprobado por la Asociación Estadounidense para la Acreditación de Cuidado de Animales de Laboratorio.INTERVENCIONES:Se colocaron esferoides en grupos de tratamiento: sin tratamiento, vehículo (sulfóxido de dimetilo) y saquinavir 15 µM. Se tomaron imágenes de los esferoides inmediatamente antes del tratamiento y 24 horas después del tratamiento.PRINCIPALES MEDIDAS DE RESULTADO:Los diámetros de los esferoides se midieron con ImageJ y se calculó el porcentaje medio de reducción de cada esferoide para determinar el efecto del tratamiento sobre el crecimiento de los esferoides. El análisis de varianza mediante comparaciones por pares se realizó con las pruebas de diferencia mínima significativa protegida de Fisher.RESULTADOS:Los grupos sin tratamiento (n =119 esferoides) y vehículo (n=126 esferoides) demostraron un aumento en el diámetro del esferoide durante el período de tratamiento. Por el contrario, los esferoides tratados con saquinavir (n =151 esferoides) demostraron una reducción porcentual estadísticamente significativa en comparación con los grupos sin tratamiento ( p < 0,0001) y con vehículo (p = 0,002).LIMITACIONES:una limitación de estos datos es que es probable que haya algún error humano dado que las imágenes fueron analizadas por tres científicos diferentes.CONCLUSIONES:Saquinavir conduce a una reducción porcentual estadísticamente significativa en el crecimiento de esferoides de tumores anales en ratones ex-vivo en comparación con los grupos de control. La terapia con inhibidores de la proteasa puede ser un tratamiento eficaz o una terapia adyuvante del protocolo Nigro para promover la regresión del tumor del cáncer anal. Consulte Video Resumen en http://links.lww.com/DCR/C82 . (Traducción-Dr. Felipe Bellolio ).

Anal Cancer Prevention Through the Topical Use of Single or Dual PI3K/mTOR Inhibitors.

INTRODUCTION: Anal dysplasia and anal cancer are major health problems. This study seeks to determine if inhibition of mTOR and/or PI3K pathways is effective at anal cancer prevention in mice with/without established precancerous lesions of the anus (anal dysplasia). METHODS: K14E6/E7 mice were entered into the study at 5 wk, 15 wk, or 25 wk of age. Mice were treated with a topical carcinogen, 7,12-Dimethylbenz[a]anthracene (DMBA), which ensures carcinoma development within 20 wk. Treatment groups included: no treatment, DMBA only, topical Pictilisib (PI3K inhibitor) with/without DMBA, topical Sapanisertib (mTOR inhibitor) with/without DMBA, and topical Samotolisib (dual PI3K/mTOR inhibitor) with/without DMBA. Mice underwent weekly observations for anal tumor development (tumor-free survival). After 20 wk of treatment, anal tissue was harvested and evaluated histologically for squamous cell carcinoma (SqCC). RESULTS: All topical treatments in conjunction with DMBA increased tumor-free survival in mice that started treatment at 15 wk of age when compared to DMBA-only treatment, except for Pictilisib + DMBA in males. Topical Sapanisertib increased tumor-free survival in mice regardless of starting treatment age. When examining tissue for microscopic evidence of SqCC, only topical Samotolisib in males decreased SqCC in the 15 wk starting mice. CONCLUSIONS: Sapanisertib, the mTOR inhibitor, had the greatest effect, in terms of increasing tumor-free survival, regardless of starting time point or sex. Unlike the other treatments, Samotolisib, the dual PI3K/mTOR inhibitor, decreased microscopic evidence of SqCC when starting treatment at 15 wk of age but only in male mice.

The use of a topical protease inhibitor, Saquinavir, to alleviate mouse papillomavirus-mediated anal disease.

Select protease inhibitors (PI) have been found to be effective in decreasing human papillomavirus oncoprotein expression. This study evaluated whether the topical PI, Saquinavir (SQV), promotes viral clearance in an infectious mouse model with Mus musculus papillomavirus 1 (MmuPV1). NOD scid gamma (NSG) mice were anally infected with ∼4 × 108 viral genome equivalents of MmuPV1 and 120 days post-infection (when majority have high-grade anal dysplasia), began topical treatments: control (mock), 7,12-dimethylbenz(a)anthracene (DMBA) only, once weekly to promote carcinogenesis, 1% SQV only, daily (Monday - Friday), and SQV + DMBA. Viral MmuPV1 load was analyzed from anal lavages pre and post-treatment. Anal tissue was harvested, processed, and evaluated for drug absorption, grade of anal disease, and anal viral RNA. Results suggest that topical SQV promotes decreased viral shedding in female mice treated with SQV.

Efficacy of Topically Administered Dihydroartemisinin in Treating Papillomavirus-Induced Anogenital Dysplasia in Preclinical Mouse Models.

The artemisinin family of compounds is cytopathic in certain cancer cell lines that are positive for human papillomaviruses (HPV) and can potentially drive the regression of dysplastic lesions. We evaluated the efficacy of topical dihydroartemisinin (DHA) on cervical dysplasia and anal dysplasia in two papillomavirus mouse models: K14E6/E7 transgenic mice, which express HPV16 oncogenes; and immunodeficient NOD/SCID gamma (NSG) mice infected with Mus musculus papillomavirus (MmuPV1). Mice started treatment with DHA at 25 weeks of age (K14E6/E7) or 20 weeks post infection (MmuPV1-infected), when the majority of mice are known to have papillomavirus-induced low- to high-grade dysplasia. Mice were treated with or without topical DHA at the cervix or anus and with or without topical treatment with the chemical carcinogen 7,12 dimethylbenz(a)anthracene (DMBA) at the anus of in transgenic mice to induce neoplastic progression. Mice were monitored for overt tumor growth, and tissue was harvested after 20 weeks of treatment and scored for severity of histological disease. For MmuPV1-infected mice, anogenital lavages were taken to monitor for viral clearance. Tissues were also evaluated for viral gene expression at the RNA and/or protein levels. Treatment with topical DHA did not reduce dysplasia in the anogenital tract in either papillomavirus-induced mouse model and did not prevent progression to anal cancer in the DMBA-treated K14E6/E7 mice.

PI3K/mTOR inhibition prevents anal cancer in mice with established low-grade anal dysplasia.

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 (4.5 mg/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 (LC3ß)), 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). LC3ß 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.

Systemic Delivery of a Dual PI3K/mTOR Inhibitor More Effective than Topical Delivery in Preventing Anal Carcinogenesis in an HPV Transgenic Mouse Model.

Introduction: Anal dysplasia is a growing health concern that over time can result in squamous cell carcinoma (SqCC) of the anus. In this study, we compare a topical versus systemic (oral) administration of LY3023414, a dual PI3K/mTOR inhibitor, to prevent anal carcinogenesis in a Human Papillomavirus (HPV) mouse model of anal cancer. Materials and Methods: K14E6/E7 transgenic mice were used to model HPV-induced anal carcinogenesis. Mice with varying starting anal histologies (normal histology, low-grade, and high-grade anal dysplasia) were treated topically at the anus or systemically via oral gavage with LY3023414 with or without topical carcinogen for 20 weeks. Mice were monitored for overt anal tumor development and anal tissue was assessed for histology and markers of PI3K and mTOR activity (pAKT and pS6, respectively). Results: LY3023414 treatment, regardless of the mode of delivery, significantly decreased overt tumor development in mice starting with normal histology and low-grade anal dysplasia. Systemic LY3023414 treatment was more effective in delaying tumor onset than topical treatment. Mice treated with systemic LY3023414 had significantly reduced rates of anal SqCC when starting with normal and low-grade anal dysplasia compared to topical treatment. Topical treatment was only effective in reducing SqCC in the setting of low-grade dysplasia. LY3023414 inhibition of pAKT and pS6 expression varied with starting histology. Neither treatment mode was effective in the setting of high-grade anal dysplasia. Conclusion: Systemic LY3023414 treatment was more effective than topical application in delaying the progression of normal anal histology and low-grade dysplasia to anal cancer in HPV-associated mice.