Mouse Squamous Cell Carcinoma Research Articles

Ceramide and sphingosine-1-phosphate act as photodynamic therapy-elicited damage-associated molecular patterns: Cell surface exposure

Molecules that appear on the surface of tumor cells after their therapy treatment may have important roles either as damage-associated molecular patterns (DAMPs) or signals for phagocytes influencing the disposal of these cells. Treatment of SCCVII and CAL27 cells, models of mouse and human squamous cell carcinoma respectively, by photodynamic therapy (PDT) resulted in the presentation of ceramide and sphingosine-1-phosphate (S1P) on the cell surface. This was documented by anti-ceramide and anti-S1P antibody staining followed by flow cytometry. The exposure of these key sphingolipid molecules on PDT-treated tumor cells was PDT dose-dependent and it varied in intensity with different photosensitizers used for PDT. The above results, together with the finding that both ceramide and S1P can activate NFκB signaling in macrophages co-incubated with PDT-treated tumor cells, establish that these two sphingolipids can act as DAMPs stimulating inflammatory/immune reactions critical for tumor therapy response.

Transdifferentiation of lung adenocarcinoma in mice with Lkb1 deficiency to squamous cell carcinoma

Lineage transition in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of non-small cell lung cancer, as implicated by clinical observation of mixed ADC and SCC pathologies in adenosquamous cell carcinoma, remains a fundamental yet unsolved question. Here we provide in vivo evidence showing the transdifferentiation of lung cancer from ADC to SCC in mice: Lkb1-deficient lung ADC progressively transdifferentiates into SCC, via a pathologically mixed mAd-SCC intermediate. We find that reduction of lysyl oxidase (Lox) in Lkb1-deficient lung ADC decreases collagen disposition and triggers extracellular matrix remodelling and upregulates p63 expression, a SCC lineage survival oncogene. Pharmacological Lox inhibition promotes the transdifferentiation, whereas ectopic Lox expression significantly inhibits this process. Notably, ADC and SCC show differential responses to Lox inhibition. Collectively, our findings demonstrate the de novo transdifferentiation of lung ADC to SCC in mice and provide mechanistic insight that may have important implications for lung cancer treatment.

Preclinical safety and activity of recombinant VSV‐IFN‐β in an immunocompetent model of squamous cell carcinoma of the head and neck

Recombinant vesicular stomatitis virus expressing interferon-β (VSV-IFN-β) has demonstrated antitumor activity in vitro and in vivo. In preparation for clinical testing in human squamous cell carcinoma (SCC) of the head and neck, we conducted preclinical studies of VSV-IFN-β in syngeneic SCC models. In vitro, VSV-IFN-β (expressing rat or mouse interferon [IFN]-β)-induced cytotoxicity and propagated in rat (FAT-7) or mouse (SCC-VII) SCC cells during normoxia and hypoxia. In vivo, intratumoral administration of VSV-rat-IFN-β or VSV-human-IFN-β in FAT-7 bearing or non-tumor bearing immunocompetent rats did not result in acute organ toxicity or death. VSV-r-IFN-β replicated predominantly in tumors and a dose dependent anti-VSV antibody response was observed. Intratumoral or intravenous administration of VSV-IFN-β resulted in growth delay and improved survival compared with controls. The above data confirm safety and feasibility of VSV-IFN-β administration in immunocompetent animals and support its clinical evaluation in advanced human head and neck cancer.

The effect of stress on efficacy of dendritic cell therapy for esophageal squamous cell carcinoma.

69 Background: Recently, abscopal effect, by which systemic anti-tumor effect by local radiation therapy (RT) was elicited, has been reported. This phenomenon was thought to be mediated by stress protein gp96, a key molecule in antigen-presenting. We tested the enhancement of dendritic cell (DC) therapy by various stress, such as radiation or modulated electro-hyperthermia (mEHT) in DC therapy for squamous cell carcinoma (SCC). Methods: Mouse SCC cells, SCCVII, were inoculated into the left femur (primary tumor) of C3H/He mice subcutaneously, and also similarly inoculated into non-treated chest subcutaneous tissue (secondary tumor). Only the primary tumor was exposed to 4 or 10 Gy of RT or 5-10 watts of mEHT. Then DCs was injected only into the primary tumor. Following that, tumor volumes of the primary and secondary tumor were measured. Next, the population of immune-related T-cells in tumor-draining lymph nodes (TDLN) and gp96 expression in the primary tumor were evaluated. Results: With intratumoral injection of DCs and RT, primary tumor growth was markedly suppressed. Furthermore, secondary tumor growth was also suppressed. On the other hand, the combination of DCs and mEHT could inhibit tumor growth at both the primary and the secondary tumor. As for the population of T-cells, the population of CD3+, CD 4+ and CD 8+T-cells in the TDLN was markedly elevated and that of FoxP3 was reduced my mEHT. Gp96 expression, a stress protein, was strongly enhanced after RT and mEHT. Conclusions: Stress, such as RT or hyperthermia can enhance the efficacy of DC therapy and can induce a strong antitumor effect by stimulating the expression of stress protein, gp96. These combination therapies can be a strong systemic immunotherapy for esophageal SCC.

Increased killing of SCCVII squamous cell carcinoma cells after the combination of Pc 4 photodynamic therapy and dasatinib is associated with enhanced caspase-3 activity and ceramide synthase 1 upregulation

Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1-and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of ceramide synthase 1 mRNA but not the production of ceramide. The data imply potential significance of the combination for cancer treatment.

Enhancement of systemic tumor immunity for squamous cell carcinoma cells by an oncolytic herpes simplex virus

RH2 is a neurovirulent γ134.5 gene-deficient herpes simplex virus type 1 (HSV-1) with a lytic ability in human squamous cell carcinoma (SCC) cells; it is related to spontaneously occurring HSV-1 mutant HF10. The effect of RH2 on SCC was examined using a syngeneic C3H mouse model. After infection of mouse SCCVII cells with RH2, cell viability was decreased at first, but recovered by prolonged culture, indicating the limited replication of RH2. The antitumor ability of RH2 was examined using a bilateral SCCVII tumor model. The growth of the RH2-injected tumors was suppressed compared with that of phosphate-buffered saline-injected tumors. Moreover, the growth of contralateral tumor of RH2-treated mice was also suppressed significantly. The splenocytes of C3H mice treated with RH2 lysed more SCCVII cells than NFSaY83 cells and YAC-1 cells. The cytotoxicity of the splenocytes on SCCVII cells was significantly greater than that of splenocytes from tumor-bearing mice. Removal of CD8(+) T cells from splenocytes decreased their cell killing activity remarkably. The antitumor effect of RH2 on SCCVII xenografts in nude mice was not demonstrated. These results indicate that RH2 exhibited a suppressive effect on mouse SCC, even if the replication of RH2 was limited. This is ascribed to the ability of RH2 to enhance existing tumor-specific cytotoxic T lymphocyte activity.

C-reactive protein inhibits lymphangiogenesis and resultant lymph node metastasis of squamous cell carcinoma in mice

Lymph node involvement is the most important prognostic factor in many solid cancers. Recently, we found that patients with esophageal and lung cancer carrying the C-reactive protein (CRP) 1846T/T genotype, which is associated with lower serum CRP levels, are more likely to have lymph node metastasis. We hypothesized that host CRP directly inhibits lymph node metastasis. We inoculated NR-S1M metastatic cells subcutaneously into the backs of C3H/HeN mice. Concurrently, 1 μg of recombinant mouse CRP or phosphate-buffered saline was injected subcutaneously every 3 days for 5 weeks, after which the mice were killed for evaluation. We evaluated lymph node metastasis and lymphangiogenesis in the implanted tumor by using immunohistochemical analysis with anti-pancytokeratin and antilymphatic vessel endothelial hyaluronan receptor-1 antibodies. There was no substantial difference in tumor size between the 2 groups but the lymph nodes were smaller in the CRP group than the control group (P < .044). Immunohistochemical analysis confirmed inguinal lymph node metastasis in 70% (14/20) of control mice, but in only 30% (3/10) of mice in the CRP group. Moreover, the metastatic area within lymph nodes was less in the CRP group (P < .042) and tumoral lymphangiogenesis was decreased in the CRP group (P < .037). CRP appears to inhibit tumoral lymphangiogenesis and lymph node metastasis in mice. These findings suggest that by inhibiting lymph node metastasis, CPR may have therapeutic potential for use against cancer.

The drug efficacy and adverse reactions in a mouse model of oral squamous cell carcinoma treated with oxaliplatin at different time points during a day

BackgroundRecent studies have shown that the growth and proliferation of cancer cells in vivo exhibit circadian rhythm, and the efficacy and adverse reactions of platinum-based anticancer drugs administered at different times of the day vary significantly on colon cancer. However, since the circadian rhythms of growth and proliferation of various cancer cells often differ, the question of whether the administration of platinum anticancer drugs at different times of the day exerts significantly different efficacy and adverse effects on oral cancers remains to be elucidated. This study has compared the efficacy and adverse effects of oxaliplatin (L-OHP) administration at different times during a day on oral squamous cell carcinoma in mice and has analyzed cellular circadian rhythms.MethodsThe mouse model for oral squamous cell carcinoma was established in 75 nude mice, housed in a 12 hour light/12 hour dark cycle environment. The mice were randomly divided into five groups; four experimental groups were intravenously injected with L-OHP at four time points within a 24-hour period (4, 10, 16, and 22 hours after lights on [HALO]). The control group was intravenously injected with the same volume of saline. Treatment efficacy and adverse reactions were compared on the seventh day after the injection, at 22 HALO. The existence of circadian rhythms was determined by cosine analysis.ResultsOnly injections of L-OHP at 16 and 22 HALO significantly prolonged animal survival time. The adverse reactions in mice injected with L-OHP at 16 and 22 HALO were significantly less than those observed in mice administered L-OHP at 4 and 10 HALO. The cosine fitting curve showed that the survival time and adverse reactions exhibited circadian rhythm.ConclusionThe time factor should be considered when treating patients with oral squamous cell carcinoma with L-OHP in order to achieve better efficacy, reduce the adverse reactions, and improve the patients’ survival time and quality of life.

Abstract 178: Vitamin D reduces lung cancer progression the N-nitroso-tris-chloroethylurea model of mouse lung squamous cell carcinoma.

Abstract Vitamin D, an essential mediator of calcium homeostasis, acts through the vitamin D receptor to promote cellular differentiation and inhibit proliferation. Epidemiologic studies indicate that low serum levels of 25(OH)D3 are associated with increased risk and poor prognosis of lung cancer. Pre-clinical studies in mice show that dietary vitamin D and calcium deficiency promote carcinogenesis in mouse models of cancer. These data suggest that vitamin D may have a role as a chemopreventive agent. To investigate the role of vitamin D in lung cancer, a carcinogen-induced model of lung squamous cell carcinoma (SCC) was employed. N-nitroso-tris-chloroethylurea (NTCU) was applied topically each week to the back of SWR/J mice. Following ∼15 weeks of NTCU treatment, mice on a standard diet begin to develop flat atypia, which progresses through dysplasia to invasive lung SCC by 35 to 40 weeks. We investigated the effects of vitamin D on cancer progression using different levels of dietary vitamin D3 and injections of the most active metabolite 1,25(OH)2D3 (calcitriol) in the NTCU model (80 mM/week). After 24 weeks of NTCU, mice on a deficient diet had more advanced disease than those on a sufficient diet. However, that study demonstrated that the 80 mM/week of NTCU was too toxic and resulted in premature death. A second vitamin D intervention study has now been completed using a less toxic dose of 40 mM/week of NTCU. In this study mice were randomized to 6 treatment groups (Table 1). Disease burden, as measured by histology (Table 1), was similar to that seen with higher dose NCTU. This study supports our earlier findings that vitamin D deficient mice develop disease at a more rapid rate when compared to mice that are sufficient. This also suggests that calcitriol administration may reduce the progression of lung SCC in mice that are vitamin D deficient. Further studies are on going to elucidate the mechanism by which vitamin D acts to slow the progression of lung SCC in this model. Table 1. Serum 25(OH)D3 levels &amp; incidence of high-grade dysplasia and lung SCC. Group Carcinogen (NTCU) Diet Vitamin D (IU/kg) Serum 25(OH)D3 Level (ng/ml) High-grade Dysplasia (%) Lung SCC (%) D deficient − 0 &amp;lt;4 0/11 (0) 0/11 (0) D deficient +NTCU + 0 &amp;lt;4 13/13 (100) 11/13 (85) D deficient +NTCU &amp; calcitriol (i.p. 80 ug/kg/w) + 0 &amp;lt;4 14/14 (100) 8/14 (57) D sufficient − 2,000 21 0/14 (0) 0/14 (0) D sufficient +NTCU + 2,000 19 13/14 (92) 4/14 (29) D sufficient +NTCU &amp; calcitriol (i.p. 80 ug/kg/w) + 2,000 10 10/13 (76) 3/13 (20) Citation Format: Sarah A. Mazzilli, Mary Reid, Paul Bogner, Donald Trump, Candace Johnson. Vitamin D reduces lung cancer progression the N-nitroso-tris-chloroethylurea model of mouse lung squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 178. doi:10.1158/1538-7445.AM2013-178

Abstract 4875: Topically applied Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin inhibits ultraviolet radiation-induced development of cutaneous squamous cell carcinomas.

Abstract Skin cancer is the second most common malignancy encountered in the U.S. with an expected diagnosis of 1.3 million new cases of non-melanoma skin cancer each year. Cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the most common non-melanoma forms of human skin cancer. SCC, unlike BCC, invades the nearby tissues. SCC is mainly caused by cumulative ultraviolet radiation (UVR) exposure over the course of a lifetime. Certain vulnerable populations, e.g., organ transplant recipients, have extraordinarily high rates of SCC (&amp;gt;50%) with markedly worse morbidity and mortality. To date, there are no accepted criteria for defining or managing these SCC patients. We found that PKCε is an important component of UVR-induced signal transduction pathways to the development of SCC. Also, PKCε is expressed in human and mouse SCC. To find clues about the mechanism by which PKCε may impart susceptibility to UVR-induced development of SCC, we found that PKCε interacts with Hsp90β. In this experiment, PKCε transgenic mice were exposed once to a UVR (4 kJ/m2). PKCε-Hsp90 interaction was analyzed, by co-immunoprecipitation and Western blotting, at 1 and 2hr post UVR treatment. PKCε- Hsp90β interaction appears to be enhanced as early as 1hr post UVR treatment. The co-localization of PKCε with Hsp90β was observed in mouse epidermis, UVR-induced SCC in SKH-1 hairless mice and human SCC. Topical application of Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG) to mice inhibited UVR-induced Hsp90β-PKCε interaction, decreased PKCε expression levels and inhibited UVR-induced development of SCC. To determine the effects of topical 17AAG on UVR-induced development of SCC, the SKH1 hairless mice (6-7 week old) were exposed to UVR. The UVR source was Kodacel- filtered FS-40 sun lamps (approximately 60% UVB and 40% UVA). Mice were exposed to UVR (1.8 kJ/m2) three times weekly (Monday, Wednesday and Friday). The mice in the vehicle group (n=18) received topical treatment of 200μl vehicle (DMSO: acetone: 1:40 v/v) before and after UVR exposures. The mice in treatment group (n=13) received freshly prepared 500nmol of 17AAG (DMSO: acetone: 1:40 v/v) before and after UVR exposure. Topical 17AAG decreased SCC latency by 17 weeks and significantly (P&amp;lt;0.001) inhibited SCC incidence. 17AAG-caused inhibition of UVR-induced SCC accompanied inhibition of UVR-induced hyperplasia, Hsp90β and PKCε expression levels. Interestingly, 17AAG-caused inhibition of UVR-induced Hsp90β accompanied an increase in Hsp70 protein level. These results led us to conclude that disruption of UVR-induced interaction of Hsp90β with PKCε and other oncogenic client proteins by Hsp90 inhibitor 17AAG may result in prevention and treatment of UVR-induced SCC. (Support: RO1CA102431). Citation Format: Anupama Singh, Samuel J. Bauer, Ashok Singh, Jordan M. Sand, Bilal B. Hafeez, Louise Meske, Ajit K. Verma. Topically applied Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin inhibits ultraviolet radiation-induced development of cutaneous squamous cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4875. doi:10.1158/1538-7445.AM2013-4875

Up-Regulation of Cyclooxygenase 2 and Matrix Metalloproteinases-2 and -9 in Cutaneous Squamous Cell Carcinoma: Active Role of Inflammation and Tissue Remodeling in Carcinogenesis

BackgroundTissue inflammation and remodeling have been extensively studied in various tumors in relation with their invasiveness and metastasis.ObjectiveThe purpose of this study was to investigate the change in tissue inflammation and remodeling markers in cutaneous squamous cell carcinoma (SCC).MethodsExpression levels of cyclooxygenase-2 (COX-2) as an inflammatory marker and matrix metalloproteinases-2 and -9 (MMPs 2/9) as remodeling markers were studied in mouse and human SCCs. Western blot analysis and RT-PCR for COX-2 and MMPs 2/9 were performed with skin samples from SCC patients and chronic ultraviolet B (UVB)-induced SCC from hairless mice.ResultsmRNA and protein levels of COX-2 and MMPs 2/9 were up-regulated with the higher sensitivity for MMP-9 in mouse SCCs, which were induced by chronic UVB irradiation. Consistently, COX-2 and MMPs 2/9 were up-regulated with the higher sensitivity for MMP-9 in human SCCs.ConclusionCOX-2 and MMPs 2/9 are up-regulated in well-differentiated cutanous SCC. Our findings indicate that inflammatory and tissue remodeling processes are actively induced during carcinogenesis of cutaneous SCC.

Non-melanoma skin cancer in mouse and man

As a frontier organ, skin is exposed to different environmental and/or occupational chemicals which cause cutaneous cancers in experimental animals. In mice, 7,12-dimethylbenz[a]anthrancene (DMBA) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) are frequently used as skin model tumor initiator and promoter, respectively. The sequential administration of DMBA and TPA leads to the appearance of a large number of benign papillomas, of which some convert later into invasive squamous cell carcinomas (SCC). At the molecular level, initiation of carcinogenesis in mouse skin consists in the mutational activation of the Ha-ras oncoprotein. HA-RAS mutations are rare in human SCC, but HA-RAS-mutated tumors appear in melanoma patients treated with B-raf inhibitors, indicating that initiated, HA-RAS-mutated stem cells also reside in human skin. Similarly, UV-induced human SCC show footprint mutations in the tumor suppressor gene TP53 which are also observed in UV-induced mouse SCC. Strong species differences exist with respect to phorbol ester-mediated tumor promotion. While certain mouse strains are very susceptible, other rodent species are much less sensitive. Likewise, humans appear to be much more resistant to phorbol ester-mediated skin toxicity. Papilloma formation as a result of a chemical insult is uncommon in men, questioning the relevance of this preneoplastic lesion for humans. However, skin tumorigenesis in the experimental situation and in humans appears to follow common molecular mechanisms, even though there are species differences in the morphological correlates to the preneoplastic state. Therefore, we recommend not simply labeling them as irrelevant for human risk assessment.

Near Infrared Imaging of EGFR of Oral Squamous Cell Carcinoma in Mice Administered Arsenic Trioxide

BackgroundThe effectiveness of near-infrared imaging (NIR) interrogation of epidermal growth factor receptor (EGFR) expression as a sensitive biomarker of oral squamous cell carcinoma (OSCC) response to arsenic trioxide therapy was studied in mice.Material and MethodsA431 OSCC in vitro were exposed to 0 µM, 0.5 µM, 2.5 µM, or 5 µM of As2O3 for 0 h, 24 h, 48 h and 72 h. Confocal microscopy and flow cytometry confirmed EGFR expression and demonstrated a sensitivity dose-related signal decline with As2O3 treatment. Next, mice with pharynx-implanted A431 cells received As2O3 i.p. every 48 h at 0.0, 0.5, 2.5, or 5 mg/kg/day (n = 6/group) from day 0 to 10. An intravenous NIR probe, EGF-Cy5.5, was injected at baseline and on days 4, 8, and 12 for dynamic NIR imaging. Tumor volume and body weights were measured three times weekly.Results In vitro, A431 EGFR expression was well appreciated in the controls and decreased (p<0.05) with increasing As2O3 dose and treatment duration. In vivo EGFR NIR tumor signal intensity decreased (p<0.05) in As2O3 treated groups versus controls from days 4 to 12, consistent with increasing dosage. Tumor volume diminished in a dose-related manner while body weight was unaffected. Immunohistochemical staining of excised tumors confirmed that EGFR expression was reduced by As2O3 treatment in a dose responsive pattern.ConclusionThis study demonstrates for the first time that OSCC can be interrogated in vivo by NIR molecular imaging of the EGFR and that this biomarker is effective for the longitudinal assessment of OSCC response to As2O3 treatment.

Cutaneous Papilloma and Squamous Cell Carcinoma Therapy Utilizing Nanosecond Pulsed Electric Fields (nsPEF)

Nanosecond pulsed electric fields (nsPEF) induce apoptotic pathways in human cancer cells. The potential therapeutic effective of nsPEF has been reported in cell lines and in xenograft animal tumor model. The present study investigated the ability of nsPEF to cause cancer cell death in vivo using carcinogen-induced animal tumor model, and the pulse duration of nsPEF was only 7 and 14 nano second (ns). An nsPEF generator as a prototype medical device was used in our studies, which is capable of delivering 7–30 nanosecond pulses at various programmable amplitudes and frequencies. Seven cutaneous squamous cell carcinoma cell lines and five other types of cancer cell lines were used to detect the effect of nsPEF in vitro. Rate of cell death in these 12 different cancer cell lines was dependent on nsPEF voltage and pulse number. To examine the effect of nsPEF in vivo, carcinogen-induced cutaneous papillomas and squamous cell carcinomas in mice were exposed to nsPEF with three pulse numbers (50, 200, and 400 pulses), two nominal electric fields (40 KV/cm and 31 KV/cm), and two pulse durations (7 ns and 14 ns). Carcinogen-induced cutaneous papillomas and squamous carcinomas were eliminated efficiently using one treatment of nsPEF with 14 ns duration pulses (33/39 = 85%), and all remaining lesions were eliminated after a 2nd treatment (6/39 = 15%). 13.5% of carcinogen-induced tumors (5 of 37) were eliminated using 7 ns duration pulses after one treatment of nsPEF. Associated with tumor lysis, expression of the anti-apoptotic proteins Bcl-xl and Bcl-2 were markedly reduced and apoptosis increased (TUNEL assay) after nsPEF treatment. nsPEF efficiently causes cell death in vitro and removes papillomas and squamous cell carcinoma in vivo from skin of mice. nsPEF has the therapeutic potential to remove human squamous carcinoma.

Effective treatment of squamous cell carcinomas with ingenol mebutate gel in immunologically intact SKH1 mice

Ingenol mebutate has recently been approved by the Federal Drug Administration (USA) as a topical treatment for actinic keratoses. Herein, we describe the efficacy of ingenol mebutate for the topical treatment of squamous cell carcinoma (SCC) using a wild-type mouse model (SKH1) and the UV-induced mouse SCC cell line, T7. Daily treatment for 2 days with 0.25 % ingenol mebutate gel produced a cure rate of 70 %, with 0 % for placebo gel. Electron microscopy revealed swelling of cancer cell mitochondria within 1 h, with disruption of the inner mitochondrial membranes evident at 6 h post treatment. Primary necrosis of cancer cells was clearly evident by 24 h. Treatment was associated with local haemorrhage and a prodigious neutrophil infiltrate, with anti-T7 antibodies also detected. This is the first report of the successful treatment of SCC tumours with ingenol mebutate gel in wild-type mice, and supports the view that ingenol mebutate induces primary necrosis and activates the immune system.Electronic supplementary materialThe online version of this article (doi:10.1007/s00403-012-1270-0) contains supplementary material, which is available to authorized users.

Differentiation-induced skin cancer suppression by FOS, p53, and TACE/ADAM17

Squamous cell carcinomas (SCCs) are heterogeneous and aggressive skin tumors for which innovative, targeted therapies are needed. Here, we identify a p53/TACE pathway that is negatively regulated by FOS and show that the FOS/p53/TACE axis suppresses SCC by inducing differentiation. We found that epidermal Fos deletion in mouse tumor models or pharmacological FOS/AP-1 inhibition in human SCC cell lines induced p53 expression. Epidermal cell differentiation and skin tumor suppression were caused by a p53-dependent transcriptional activation of the metalloprotease TACE/ADAM17 (TNF-α-converting enzyme), a previously unknown p53 target gene that was required for NOTCH1 activation. Although half of cutaneous human SCCs display p53-inactivating mutations, restoring p53/TACE activity in mouse and human skin SCCs induced tumor cell differentiation independently of the p53 status. We propose FOS/AP-1 inhibition or p53/TACE reactivating strategies as differentiation-inducing therapies for SCCs.

Role of AMPK in UVB-induced DNA damage repair and growth control

Skin cancer is the most common cancer in the U.S., while DNA-damaging UVB radiation from the sun remains the major environmental risk factor. Reducing skin cancer incidence is becoming an urgent issue. The energy-sensing enzyme 5’-AMP-activated protein kinase (AMPK) plays a key role in the regulation of cellular lipid and protein metabolism in response to stimuli such as exercise and changes in fuel availability. However, the role AMPK in the response of skin cells to UVB damage and in skin cancer prevention remains unknown. Here we show that AMPK activation is reduced in human and mouse squamous cell carcinoma as compared with normal skin, and by UVB irradiation, suggesting that AMPK is a tumor suppressor. At the molecular level, AMPK deletion reduced the expression of the DNA repair protein xeroderma pigmentosum C (XPC) and UVB-induced DNA repair. AMPK activation by its activators AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) and metformin (N’,N’-dimethylbiguanide), the most widely used anti-diabetic drug, increased the expression of XPC expression and UVB-induced DNA repair in mouse skin, normal human epidermal keratinocytes, and AMPK wild-type cells but not in AMPK deficient cells, indicating an AMPK-dependent mechanism. Topical treatment with AICAR and metformin not only delayed onset of UVB-induced skin tumorigenesis but also reduced tumor multiplicity. Furthermore, AMPK deletion increased ERK activation and cell proliferation, while AICAR and metformin inhibited ERK activation and cell proliferation in keratinocytes, mouse skin, AMPK wild-type and AMPK deficient cells, suggesting an AMPK-independent mechanism. Finally, in UVB-damaged tumor-bearing mice, both topical and systemic metformin prevented the formation of new tumors and suppressed growth of established tumors. Our findings not only suggest that AMPK is a tumor suppressor in the skin by promoting DNA repair and controlling cell proliferation, but also demonstrate previously unknown mechanisms by which the AMPK activators prevent UVB-induced skin tumorigenesis.

Abstract SY10-01: Uncovering the cellular origin of skin cancers

Abstract Stem cells (SCs) are responsible for tissue homeostasis and repair. SCs have been hypothesized to be the cells at the origin of cancer. However, for the vast majority of cancers, the cell at the origin of tumor initiation is still unknown. Two epithelial skin cancers are frequent in human populations: the basal cell carcinoma (BCC) and the squamous cell carcinoma (SCC). We developed new genetic approaches to identify the cells at the origin of BCC and SCC in mice. Using mice conditionally expressing constitutively active Smoothened mutant (SmoM2) or KRas mutant (G12D), we activated hedgehog or Ras signaling in different cellular compartments of the skin epidermis, and determined from which epidermal compartments oncogene expression induces cancer formation. Using clonal analysis, we found that SmoM2 induced BCC arise from long-term resident progenitor cells of the interfollicular epidermis. In contrast, expression of mutant KRas in hair follicle bulge stem cells (SCs), but not in their transient amplifying progeny, led to benign squamous skin tumor (papilloma). Whereas no malignant tumor was observed following KRasG12D expression alone, expression of KRasG12D combined with the loss of p53 induced invasive SCC formation (3). Our studies uncover the cells at the origin of SmoM2 induced BCC and and Kras induced SCC in mice and demonstrate that expression of differentiation markers in tumor cells is not necessary predictive of the cancer initiating cells. We have now developed novel strategy to isolate by flow cytometry oncogene targeted cells at different time following oncogene expression and determined the molecular changes associated with tumor initiation and malignant progression. The functional relevance of these genes is currently investigated using gain and loss of function in mice. These new results and the relevance of these findings to human skin cancers will be presented. Acknowledgement This work is supported by Welbio, the FNRS, the program d'excellence CIBLES of the Wallonia Region, Fondation Contre le Cancer, the European Research Council (ERC) and the EMBO Young Investigator Program. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY10-01. doi:1538-7445.AM2012-SY10-01

Abstract LB-65: Protein kinase Cε, which is linked to development of squamous cell carcinomas, potentiates ultraviolet radiation-induced expression of novel transcription factors GATA and PAX, which regulates expression of cell survival genes

Abstract Protein kinase C epsilon (PKCε) is among the six PKC isoforms (α, δ, ε, η, μ, ξ) expressed in both mouse and human skin. We have reported that epidermal PKCε levels dictate the susceptibility of PKCε transgenic mice (TG) to the development of squamous cell carcinomas (SCC) elicited by repeated exposures to ultraviolet radiation (UVR). Histologically, SCC in TG mice, like human SCC, is poorly differentiated and metastatic. To find clues about the mechanism by which PKCε may impart susceptibility to UVR-induced development of SCC, we compared the effects of UVR treatment of TG mice with their wildtype (WT) littermates on global transcription factors (TFs) activation profile. In this experiment, mice were exposed to UVR (2kj/m2) four times (Monday, Wednesday, Friday and Monday). At 1hr and 3hrs post last UVR exposure, these mice were sacrificed. Nuclear lysates were prepared from the scraped epidermis of these mice. To screen for unknown TFs, we employed the Panomics Combo Protein array, which contains consensus complimentary sequences of 345 TFs binding sites. Array results from samples of UVR exposed mice were directly compared to the unexposed mice. TFs that had increased over 2-fold in the UVR exposure experiments were scored as positive for activation. We observed (&amp;gt;2-fold) activation of 70 TFs as early as 1hr post UVR exposure. Among all seventy TFs, highly activated TFs were GATA-1, GATA-2, PAX-4, PAX-6, PAX-8, Thy-1BP, HFH-2, HFH-3, HIF-1, CEA, HOXD8/9/10, AhR/Amt, and MUSF. We performed the validation of various activated TFs and among them we found that GATA and PAX families of transcription factors were found activated. UVR-induced expression level of GATA-1, GATA-2, PAX-4 was enhanced in PKCε over-expressing transgenic mice as compared to wild-type littermates. We employed the Genego program and found that VEGFR-2, CyclinD1, Bcl-2, c-Myc, BMP-4, WT1 and survivin are the downstream target genes of GATA1 and GATA-2. All of these genes are known to play role in the cell survival pathway. We have previously shown that PKCε overexpressing transgenic mice also exhibit constitutive activation of Stat3. Taken together, we conclude that PKCε-mediated activation of Stat-3, GATA-1, GATA-2 and PAX-4 may contribute to the susceptibility of PKCε transgenic mice to UVR-induced development of SCC. (Support: NIH Grant CA 35368) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-65. doi:1538-7445.AM2012-LB-65

Abstract 2560: IKKα inactivation predisposes to spontaneous lung squamous cell carcinomas

Abstract Lung cancer is the leading cause of cancer deaths worldwide. To advance the understanding of this disease, various genetically engineered and chemical induced mouse models have been established. However, most animal models resemble human lung adenocarcinoma, and spontaneous lung squamous cell carcinomas (SCCs) mouse models are very rare. Here, we generated Ikkα-KA/KA knock-in mice (KA/KA) in which an ATP binding site of IKKα, Lys 44 was replaced by alanine. The knock-in mice develop severe skin lesions and begin to die after 6 months. We found lung SCCs in some of the mice. To study lung SCC development, we decided to stabilize the skin condition by reintroducing transgenic IKKα by crossing KA/KA with Lori.IKKα transgenic mice to generate KA/KA/Lori.IKKα (KA/KAL) mice. Almost all the KA/KAL (100%) mice at 4 to 6 months of age developed spontaneous lethal lung SCCs. The endogenous IKKα protein level generally markedly declined in an age dependent manner in these IKKα mutant mice. Progenitor cell related markers Sox2, OCT3/4 and Nanog were only increased in the lung SCC tissue but not in the tumor adjacent tissues, implying the involvement of cancer stem cells in lung SCC. Furthermore, we detected substantial increases in Ras and CyclinD1 levels and EGFR, ERK and p38 activities in lung SCCs. On the other hand, we detected reduction in tumor suppressor gene Rb and IαBβ accompanying with reduced IKKα levels in KA/KAL lungs as well as in lung SCCs. Importantly, we observed a similar alteration pattern in mouse and human lung SCCs. Finally, reintroducing IKKα into lung epithelial cells prevented lung SCC development in mice. Collectively, our study supports the tumor suppressing role of IKKα in lung tumorigenesis. This novel lung SCC mouse model may facilitate investigations in pathogenesis, diagnosis, and treatment of human lung SCC disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2560. doi:1538-7445.AM2012-2560