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Supplementary Figure 1 from All-Trans Retinoic Acid Suppresses Stat3 Signaling during Skin Carcinogenesis

Supplementary Table 1 from Identification of Genes Correlated with Early-Stage Bladder Cancer Progression

Supplementary Figure 3 from All-Trans Retinoic Acid Suppresses Stat3 Signaling during Skin Carcinogenesis

Transitional cell carcinoma (TCC) of the bladder ranks fourth in incidence of all cancers in the developed world, yet the mechanisms of its origin and progression remain poorly understood. There are also few useful diagnostic or prognostic biomarkers for this disease. We have combined a transgenic mouse model for invasive bladder cancer (UPII-SV40Tag mice) with DNA microarray technology to determine molecular mechanisms involved in early TCC development and to identify new biomarkers for detection, diagnosis, and prognosis of TCC. We have identified genes that are differentially expressed between the bladders of UPII-SV40Tag mice and their age-matched wild-type littermates at 3, 6, 20, and 30 weeks of age. These are ages that correspond to premalignant, carcinoma in situ, and early-stage and later stage invasive TCC, respectively. Our preliminary analysis of the microarray data sets has revealed ∼1,900 unique genes differentially expressed (≥3-fold difference at one or more time points) between wild-type and UPII-SV40Tag urothelium during the time course of tumor development. Among these, there were a high proportion of cell cycle regulatory genes and a proliferation signaling genes that are more strongly expressed in the UPII-SV40Tag bladder urothelium. We show that several of the genes upregulated in UPII-SV40Tag urothelium, including RacGAP1, PCNA, and Hmmr, are expressed at high levels in superficial bladder TCC patient samples. These findings provide insight into the earliest events in the development of bladder TCC as well as identify several promising early-stage biomarkers. Cancer Prev Res; 3(6); 776–86. ©2010 AACR.

Supplementary Figure 2 from All-Trans Retinoic Acid Suppresses Stat3 Signaling during Skin Carcinogenesis

Supplementary Table 2 from Identification of Genes Correlated with Early-Stage Bladder Cancer Progression

Related Articles from The Promise of Natural Products for Blocking Early Events in Skin Carcinogenesis

Bladder Cancer (BC) is the second common genitourinary cancer with high recurrence and mortality rate due to metastatic muscle invasive BC (MIBC). Since majority of BCs are non-invasive at diagnosis, developing agents that effectively block BC progression may be beneficial for clinical translation. In this study, clinically approved agents, everolimus, mTOR inhibitor, [0.19mg/kg;7x/week (low dose, LD) or 1.33mg/kg;1x or 2X/week (high dose, HD)] at various dosing schedules alone or in combination with naproxen, an NSAID (30mg/kg body weight) intermittent dosing (3 Wks ON/OFF) were tested for efficacy in an N-butyl-N-(4- hydroxybutyl)nitrosamine (BBN)-induced BC rat model. Female F344 rats (8 weeks age; N=30) were gavaged 16 doses of BBN (150mg/dose). Either one-week (early intervention) or 12-weeks (late intervention) after BBN treatment, rats in each group received respective drug treatments by gavage. At 50 weeks age, rats were euthanized, and tissues were analyzed. Results suggest that BBN-exposed rats developed high number of Non-MIBC (NMIBC) and MIBC and had significantly large bladders (430±57mg, Mean±SEM; p200mg) incidence by 90% (p72% reduction in tumor weight (120.8±7.8mg, 121.8±11.5mg, and 118.6±5.2mg; p Citation Format: Venkateshwar Madka, Gopal Pathuri, Surya P. Singh, Anil Singh, Anh Bao, Nicole Stratton, Stanley Lightfoot, Clinton J. Grubbs, Jennifer Fox, John L. Clifford, Brian Cholewa, Shizuko Sei, CV Rao. Chemopreventive efficacy of everolimus and naproxen combination against carcinogen induced bladder cancer in F344 rats. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5260.

Introduction A mass exposure to irradiation would be a challenge to health care systems. A simple tool or test that indicates the intensity of the absorbed radiation or the chances of survivability would be invaluable in this scenario. To identify biomarkers that potentially could provide novel biodosimetry tools, we have conducted a radiation dose-response and time-course experiment in mice that includes an assessment of the transcriptome of the skin. Methods Groups of mice (n=5) received whole-body X-ray exposures (0, 1, 3, 6, or 20Gy) and skin biopsies were obtained from each animal at times post-irradiation (h2, Days 4, 7, 21, 28). Biopsies were collected from the 20Gy cohort for only days 0, 4, and 7. Total RNA was isolated and microarrays were performed and analyzed using custom R scripts to obtain lists of probe sets differentially expressed. Changes in gene expression at Benjamini-Hochberg FDR adjusted P < 0.05 and FC >2 were deemed significant. Analyses were performed comparing the different doses of X-ray exposure over all time points. Results Mice in the 20Gy group were euthanized by d7 and the dose was considered lethal. Animals in 1, 3, and 6Gy groups completed the full experiment to d28. Sammon plot analysis of transcriptomes showed clear separation of samples based on the irradiation levels and time after exposure. The clearest separation was between samples of lethal and sublethal doses. Samples from animals exposed to sublethal doses separated more based on timepoints rather than the IR dose suggesting a level of similarity in the progression of the response to sublethal doses. Downregulation was the dominant modulation in the significantly differentially transcribed genes (SDTGs) in the 20Gy group. Temporal changes in ratios of upregulated/downregulated SDTGs (P < 0.05 and FC > 2) revealed further the difference between of transcriptome responses after exposure to lethal and sublethal doses and indicated a delayed peaks response with increasing IR doses within the sublethal range. About 59% of the SDTGs in 20gy were common to all timepoints while no more than 11% were common at the same duration in the other groups. Ratios of the number of SDTGs at h2 to those common to all TPs decreased in a dose-dependent manner with potential radiation dosimetric applications. Conclusions These results demonstrate a solid ability in detecting IR exposure, differentiating lethal and sublethal exposures, and differentiating among the exposure to sublethal doses.

Opioid-based analgesics provide the mainstay for attenuating burn pain, but they have a myriad of side effects including respiratory depression, nausea, impaired gastrointestinal motility, sedation, dependence, physiologic tolerance, and opioid-induced hyperalgesia. To test and develop novel analgesics, validated burn-relevant animal models of pain are indispensable. Herein we review such animal models, which are mostly limited to rodent models of burn-induced, inflammatory, and neuropathic pain. The latter two are pain syndromes that provide insight into the pain caused by systemic pro-inflammatory cytokines and direct injury to nerves (e.g., after severe burn), respectively. To date, no single animal model optimally mimics the complex pathophysiology and pain that a human burn patient experiences. No currently available burn-pain model examines effects of pharmacological intervention on wound healing. As cornerstones of pain and wound healing, pro-inflammatory mediators may be utilized for insight into both processes. Moreover, common clinical concerns such as systemic inflammatory response syndrome and multiple organ dysfunction remain unaddressed. For development of analgesics, these aberrations can significantly alter the potential efficacy and/or adverse effects of a prescribed analgesic following burn trauma. We therefore suggest that a multi-model strategy would be the most clinically relevant when evaluating novel analgesics for use in burn patients.

In the event of a mass casualty radiation scenario, rapid assessment of patients' health and triage is required for optimal resource utilization. Identifying the level and extent of exposure as well as prioritization of care is extremely challenging under such disaster conditions. Blood-based biomarkers, such as RNA integrity numbers (RIN), could help healthcare personnel quickly and efficiently determine the extent and effect of multiple injuries on patients' health. Evaluation of the effect of different radiation doses, alone or in combination with burn injury, on total RNA integrity over multiple time points was performed. Total RNA integrity was tallied in blood samples for potential application as a marker of radiation exposure and survival. Groups of aged mice (3-6 mice/group, 13-18 months old) received 0.5, 1, 5, 10 or 20 Gy ionizing radiation. Two additional mouse groups received low-dose irradiation (0.5 or 1 Gy) with a 15% total body surface area (TBSA) burn injury. Animals were euthanized at 2 or 12 h and at day 1, 2, 3, 7 or 14 postirradiation, or when injury-mediated mortality occurred. Total RNA was isolated from blood. The quality of RNA was evaluated and RNA RIN were obtained. Analysis of RIN indicated that blood showed the clearest radiation effect. There was a time- and radiation-dose-dependent reduction in RIN that was first detectable at 12 h postirradiation for all doses in animals receiving irradiation alone. This effect was reversible in lower-dose groups (i.e., 0.5, 1 and 5 Gy) that survived to the end of the study (14 days). In contrast, the effect persisted for 10 and 20 Gy groups, which showed suppression of RIN values4.5 with high mortalities. Radiation doses of 20 Gy were lethal and required euthanasia by day 6. A low RIN (2.5) at any time point was associated with 100% mortality. Combined radiation-burn injury produced significantly increased mortality such that no dually-injured animals survived beyond day 3, and no radiation dose1 Gy resulted in survival past day 1. More modest suppression of RIN was observed in the surviving dually challenged mice, and no statistically significant changes were identified in RIN values of burn-only mice at any time point. In this study of an animal model, a proof of concept is presented for a simple and accurate method of assessing radiation dose exposure in blood which potentially predicts lethality. RIN assessment of blood-derived RNA could form the basis for a clinical decision-support tool to guide healthcare providers under the strenuous conditions of a radiation-based mass casualty event.

Research into potentially novel biomarkers for chronic pain development is lacking. microRNAs (miRNAs) are attractive candidates as biomarkers due to their conservation across species, stability in liquid biopsies, and variation that corresponds to a pathologic state. miRNAs can be sorted into extracellular vesicles (EVs) within the cell and released from the site of injury. EVs transfer cargo molecules between cells thus affecting key intercellular signaling pathways. The focus of this study was to determine the plasma derived EV miRNA content in a chronic neuropathic pain rat model. This was accomplished by performing either spinal nerve ligation (SNL; n = 6) or sham (n = 6) surgery on anesthetized male Sprague-Dawley rats. Mechanosensitivity was assessed and plasma derived EV RNA was isolated at baseline (BL), day 3, and 15 postnerve injury. EV extracted small RNA was sequenced followed by differentially expressed (DE) miRNAs and gene target enrichment/signaling pathway analysis performed using R packages and TargetScan/Ingenuity pathway analysis (IPA), respectively. Seven of the DE miRNAs were validated by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR). The data indicated that SNL rats displayed a time-dependent threshold reduction in response to evoked stimuli from day 3 to day 15 postnerve injury. The data also revealed that 22 and 74 miRNAs at day 3 and 15, respectively, and 33 miRNAs at both day 3 and 15 were uniquely DE between the SNL and sham groups. The key findings from this proposal include (1) the majority of the DE EV miRNAs, which normally function to suppress inflammation, were downregulated, and (2) several of the plasma derived DE EV miRNAs reflect previously observed changes in the injured L5 nerve. The plasma derived DE EV miRNAs regulate processes important in the development and maintenance of neuropathic pain states and potentially serve as key regulators, biomarkers, and targets in the progression and treatment of chronic neuropathic pain. Perspective This article describes the DE miRNA content of plasma derived EVs, comparing neuropathic pain to normal conditions. This data indicates that EV miRNAs may be important in nociception and may also serve as biomarkers for chronic pain. These results encourage further research on EV miRNAs in chronic neuropathic pain sufferers.

Burn injuries have been identified as the primary cause of injury in 5% of U.S. military personnel evacuated from Operations Iraqi Freedom and Enduring Freedom. Severe burn-associated pain is typically treated with opioids such as fentanyl, morphine, and methadone. Side effects of opioids include respiratory depression, cardiac depression, decrease in motor and cognitive function, as well as the development of hyperalgesia, tolerance and dependence. These effects have led us to search for novel analgesics for the treatment of burn-associated pain in wounded combat service members. Tetrodotoxin (TTX) is a selective voltage-gated sodium channel blocker currently in clinical trials as an analgesic. A phase 3 clinical trial for cancer-related pain has been completed and phase 3 clinical trials on chemotherapy-induced neuropathic pain are planned. It has also been shown in mice to inhibit the development of chemotherapy-induced neuropathic pain. TTX was originally identified as a neurotoxin in marine animals but has now been shown to be safe in humans at therapeutic doses. The antinociceptive effects of TTX are thought to be due to inhibition of Na+ ion influx required for initiation and conduction of nociceptive impulses. One TTX sensitive sodium channel, Nav1.7, has been shown to be essential in lowering the heat pain threshold after burn injuries. To date, the analgesic effect of TTX has not been tested in burn-associated pain. Male Sprague-Dawley rats were subjected to a full thickness thermal injury on the right hind paw. TTX (8μg/kg) was administered once a day systemically by subcutaneous injection beginning 3 days post thermal injury and continued through 7 days post thermal injury. Thermal hyperalgesia and mechanical allodynia were assessed 60 and 120min post injection on each day of TTX treatment. TTX significantly reduced thermal hyperalgesia at all days tested and had a less robust, but statistically significant suppressive effect on mechanical allodynia. These results suggest that systemic TTX may be an effective, rapidly acting analgesic for battlefield burn injuries and has the potential for replacing or reducing the need for opioid analgesics.

Interleukin 6 (IL-6) has a critical role in pain mechanisms. IL-6 signals through the Janus-activated kinases 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) pathway. The contribution of JAK2 signaling in inflammation-induced hyperalgesia has not been addressed previously. The role of this pathway was investigated using the JAK2 inhibitor, AG490, in a rat model of inflammatory pain. Unilateral hind paw inflammatory pain was induced in male Sprague-Dawley rats by intraplantar (i.pl.) injection of 3.5% ʎ-carrageenan. Inflamed rats received an i.pl. injection of either 3.5% of dimethylsulfoxide or AG490 (1–10 µg). The antinociceptive effects of AG490 were assessed by 2 pain behavioral assays 4 h later: The thermal and mechanical hyperalgesia tests. AG490 (1–10 µg) significantly attenuated ʎ-carrageenan-induced thermal hyperalgesia in a dose-dependent manner. AG490 also reduced mechanical hyperalgesia. Co-administration of opioid receptor antagonist naloxone (10 µg) and AG490 (10 µg) did not reverse AG490-produced antinociceptive activity, suggesting that the µ-opioid receptor is not responsible for the anti-hyperalgesic effects of AG490. Therefore, we suggest that AG490 produces these effects by blocking JAK2 signaling. In conclusion, JAK2 inhibitors may represent a novel class of non-narcotic drugs to treat inflammatory pain.

Opioids are extensively used as analgesics to control burn pain. However, systemic administration of opioids induces multiple adverse effects that are primarily CNS mediated. Alternately, topical application of low dose of opioids directly at the site of injury could attenuate pain while avoiding CNS-mediated side effects. Pluronic lecithin organogels (PLO) have been extensively used as vehicles to deliver topical drugs. In this study, we for the first time assessed the analgesic efficacy of three opioid-PLO formulations (fentanyl, methadone & morphine) in a rat full-thickness thermal injury (FTTI) pain model. Experiments were performed using 44 adult male Sprague-Dawley rats. A single 0.1mL topical application of either morphine (5mg/mL, n=6), fentanyl (10μg/mL, n=8), methadone gel (5mg/mL, n=8), ketamine (50mg/mL, n=6), saline (0.1mL, n=8) or PLO gel alone (0.1mL, n=8) was administered to the plantar surface of the injured hindpaw on days 4 and 7 following thermal injury. The anti-hyperalgesic effects were then measured (5, 15, 30, 60 and 120min post-drug application) using the Hargreaves' thermal test. All three opioids produced statistically significant increases in paw withdrawal latency (PWL), taken as a measure of anti-hyperalgesia, in comparison to saline-treated group (P

Skin squamous cell carcinoma (SCC), the most common cancer in the USA, is a growing problem with the use of tanning booths causing sun-damaged skin. Antiproliferative effects of curcumin were demonstrated in an aggressive skin cancer cell line SRB12-p9 (P<0.05compared to control). Topical formulation was as effective as oral curcumin at suppressing tumor growth in a mouse skin cancer model. Curcumin at 15 mg administered by oral, topical, or combined formulation significantly reduced tumor growth compared to control (P=0.004). Inhibition of pAKT, pS6, p-4EBP1, pSTAT3, and pERK1/2 was noted in SRB12-p9 cells post-curcumin treatment compared to control (P<0.05). Inhibition of pSTAT3 and pERK1/2 was also noted in curcumin-treated groupsin vivo. IHC analysis revealed human tumor specimens that expressed significantly more activated pERK (P=0.006) and pS6 (P<0.0001) than normal skin samples. This is the first study to compare topical curcumin to oral curcumin. Our data supports the use of curcumin as a chemopreventive for skin SCC where condemned skin is a significant problem. Prevention strategies offer the best hope of future health care costs in a disease that is increasing in incidence due to increased sun exposure.

Bladder problems clinically present early in life as birth defects that often lead to kidney failure and late in life as overactive bladder, incontinence and related disorders. We investigated the transcriptome of mouse bladder mucosa at juvenile and adult stages by microarray to identify the pathways associated with normal, healthy growth and maturation. We hypothesized that understanding these pathways could be key to achieving bladder regeneration or reawakening normal function in the elderly population.RNA was isolated from the mucosa at 3, 6, 20 and 30 weeks postnatally. Affymetrix® Mouse 430 v2 arrays were used to profile the expression of approximately 45,000 genes. The software program Statistical Analysis of Microarrays was used to identify genes that significantly changed during the time course.No genes were significantly up-regulated during maturation. However, 66 well annotated genes demonstrated a statistically significant downward trend, of which 10 of 10 were confirmed by quantitative polymerase chain reaction. The main functions affected by age were transcription, regulation of cellular processes, neurogenesis, blood vessel development and cell differentiation. Notable genes included collagens, Mmp2, SPARC and several transcription factors, including Crebbp, Runx1, Klf9, Mef2c, Nrp1, Pex1 and Tcf4. These molecules were indirectly regulated by inferred Tgfb1 and Egf growth factors. Analysis of gene promoter regions for overrepresented upstream transcription factor binding sites identified specificity protein 1 and epidermal growth factor receptor-specific transcription factor as potentially major transcriptional regulators driving maturation related changes.These findings identify a coherent set of genes that appear to be down-regulated during urothelial maturation. These genes may represent an attractive target for bladder regeneration or for treating age related loss of function.

Integrin α6β4 is the receptor for the laminin family of extracellular matrix proteins and is widely expressed in most epithelial tissues and Schwann cells. The expression of this integrin is up-regulated in most epithelial tumors, suggesting the role of α6β4 in their progression. The tumor microenvironment is also known to enhance the signaling competence of α6β4 through functional and physical interactions with other receptors. In this review, we discuss the biological mechanisms by which integrin α6β4 promotes carcinoma cell motility and invasion that leads to mammary tumor progression.

Dextran sodium sulfate (DSS)-induced colitis is widely used to study pathological mechanisms and potential treatments of inflammatory bowel disease. Because temporal changes in genome expression profiles remain unknown in this model, we performed whole genome expression profile analysis during the development of DSS colitis in comparison with ulcerative colitis (UC) specimens to identify novel and common responses during disease. Colon tissue from DSS-treated mice was collected at days 0, 2, 4, and 6. Half of each specimen was used for histopathological analysis and half for Affymetrix whole genome expression profiling and qRT-PCR validation. Genesifter and Ingenuity software analysis was used to identify differentially expressed genes and perform interactive network analysis. Identified DSS-associated genes in mice were also compared with UC patient data. We identified 1,609 genes that were significantly altered during DSS colitis; the majority were functionally related to inflammation, angiogenesis, metabolism, biological adhesion, cellular growth and proliferation, and cell-to-cell signaling responses. Five hundred and one genes were progressively upregulated, while one hundred seventy-three genes were progressively downregulated. Changes in gene expression were validated in a subset of 33 genes by qRT-PCR, with r2 = 0.925. Ingenuity gene interaction network analysis revealed novel relationships among antigen presentation, cell morphology, and other biological functions in the DSS mouse. Finally, DSS colitis gene array data were compared with UC patient array data: 152 genes were similarly upregulated, and 22 genes were downregulated. Temporal genomewide expression profile analysis of DSS-induced colitis revealed novel associations with various immune responses and tissue remodeling events such as angiogenesis similar to those in UC patients. This study provides a comprehensive view of DSS colitis changes in colon gene expression and identifies common molecules with clinical specimens that are interesting targets for further investigation.

Transitional cell carcinoma (TCC) of the bladder ranks fourth in incidence of all cancers in the developed world, yet the mechanisms of its origin and progression remain poorly understood. There are also few useful diagnostic or prognostic biomarkers for this disease. We have combined a transgenic mouse model for invasive bladder cancer (UPII-SV40Tag mice) with DNA microarray technology to determine molecular mechanisms involved in early TCC development and to identify new biomarkers for detection, diagnosis, and prognosis of TCC. We have identified genes that are differentially expressed between the bladders of UPII-SV40Tag mice and their age-matched wild-type littermates at 3, 6, 20, and 30 weeks of age. These are ages that correspond to premalignant, carcinoma in situ, and early-stage and later stage invasive TCC, respectively. Our preliminary analysis of the microarray data sets has revealed ∼1,900 unique genes differentially expressed (≥3-fold difference at one or more time points) between wild-type and UPII-SV40Tag urothelium during the time course of tumor development. Among these, there were a high proportion of cell cycle regulatory genes and a proliferation signaling genes that are more strongly expressed in the UPII-SV40Tag bladder urothelium. We show that several of the genes upregulated in UPII-SV40Tag urothelium, including RacGAP1, PCNA, and Hmmr, are expressed at high levels in superficial bladder TCC patient samples. These findings provide insight into the earliest events in the development of bladder TCC as well as identify several promising early-stage biomarkers. Cancer Prev Res; 3(6); 776–86. ©2010 AACR.

This review explored the mechanism of breast carcinoma progression by focusing on integrins and receptor tyrosine kinases (or growth factor receptors). While the primary role of integrins was previously thought to be solely as mediators of adhesive interactions between cells and extracellular matrices, it is now believed that integrins also regulate signaling pathways that control cancer cell growth, survival, and invasion. A large body of evidence suggests that the cooperation between integrin and receptor tyrosine kinase signaling regulates certain signaling functions that are important for cancer progression. Recent developments on the crosstalk between integrins and receptor tyrosine kinases, and its implication in mammary tumor progression, are discussed. [BMB reports 2010; 43(5): 311-318]

Squamous cell carcinoma (SCC) of the skin is the most clinically aggressive form of nonmelanoma skin cancer. We have determined the effects of all-trans retinoic acid (ATRA), a naturally occurring chemopreventive retinoid, on signal transducer and activator of transcription 3 (Stat3) signaling during the development of skin SCC. Stat3 is a transcription factor that plays a critical role in cell proliferation and survival, and it is constitutively active in several malignant cell types. We have previously shown that Stat3 is required for the initiation, promotion, and progression of skin SCC. ATRA is a highly efficient suppressor of tumor formation in the two-stage mouse skin carcinogenesis model and we have shown that this effect correlates with the suppression of the B-Raf/Mek/Erk signaling pathway. In this study, we have determined the pattern of Stat3 phosphorylation throughout the course of the two-stage protocol, both in the presence and absence of ATRA. We have used both SENCAR mice and K5.Stat3C transgenic mice, which express the Stat3C protein, a constitutively active form of Stat3, in the skin. Using Western blotting and immunohistochemical staining with phosphospecific antibodies, we show that coadministration of ATRA suppressed the 12-O-tetradecanoylphorbol-13-acetate–induced phosphorylation of Stat3 in both models, but was only able to suppress tumor formation in the SENCAR mice. Surprisingly, ATRA actually enhanced tumor formation in 12-O-tetradecanoylphorbol-13-acetate–treated K5.Stat3C mice. We hypothesize that ATRA blocks tumor formation, at least in part, by targeting events upstream of Stat3, such as the B-Raf/Mek/Erk pathway, and that in the K5.Stat3C mice, in which Stat3 activity is constitutive, it cannot suppress tumor formation.

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a variety of human malignancies and has been suggested to play an important role in carcinogenesis. Recently, our laboratory demonstrated that Stat3 is required for the development of skin tumors via two-stage carcinogenesis using skin-specific loss-of-function transgenic mice. To investigate further the role of Stat3 in each stage of chemical carcinogenesis in mouse skin, i.e. initiation and promotion stages, we generated inducible Stat3-deficient mice (K5.Cre-ER(T2) x Stat3(fl/fl)) that show epidermal-specific disruption of Stat3 following topical treatment with 4-hydroxytamoxifen (TM). The epidermis of inducible Stat3-deficient mice treated with TM showed a significant increase in apoptosis induced by 7,12-dimethylbenz[a]anthracene (DMBA) and reduced proliferation following exposure to 12-O-tetradecanoylphorbol-13-acetate. In two-stage skin carcinogenesis assays, inducible Stat3-deficient mice treated with TM during the promotion stage showed a significant delay of tumor development and a significantly reduced number of tumors compared with control groups. Inducible Stat3-deficient mice treated with TM before initiation with DMBA also showed a significant delay in tumor development and a significantly reduced number of tumors compared with control groups. Finally, treatment of inducible Stat3-deficient mice that had existing skin tumors generated by the two-stage carcinogenesis protocol with TM (by intraperitoneal injection) led to inhibition of tumor growth compared with tumors formed in control groups. Collectively, these results directly demonstrate that Stat3 is required for skin tumor development during both the initiation and promotion stages of skin carcinogenesis in vivo.

Purpose: The study aims to evaluate the efficacy and toxicity of fenretinide in preventing tumor recurrence in patients with transitional cell carcinoma (TCC) of the bladder. Experimental Design: We conducted a multicenter phase III, randomized, placebo-controlled trial of fenretinide (200 mg/day orally for 12 months) in patients with non–muscle-invasive bladder TCC (stages Ta, Tis, or T1) after transurethral resection with or without adjuvant intravesical Bacillus Calmette-Guerin (BCG). Patients received cystoscopic evaluation and bladder cytology every 3 months during the 1-year on study drug and a final evaluation at 15 months. The primary endpoint was time to recurrence. Results: A total of 149 patients were enrolled; 137 were evaluable for recurrence. The risk of recurrence was considered to be “low” in 72% (no prior BCG) and intermediate or high in 32% (prior BCG) of the evaluable patients. Of the lower-risk group, 68% had solitary tumors and 32% had multifocal, low-grade papillary (Ta, grade 1 or grade 2) tumors. The 1-year recurrence rates by Kaplan-Meier estimate were 32.3% (placebo) versus 31.5% (fenretinide; P = 0.88 log-rank test). Fenretinide was well tolerated and had no unexpected toxic effects; only elevated serum triglyceride levels were significantly more frequent on fenretinide (versus placebo). The Data Safety and Monitoring Board recommended study closure at 149 patients (before reaching the accrual goal of 160 patients) because an interim review of the data showed a low likelihood of detecting a difference between the two arms, even if the original accrual goal was met. Conclusions: Although well tolerated, fenretinide did not reduce the time-to-recurrence in patients with Ta, T1, or Tis TCC of the bladder.

Lung cancers develop via multiple genetic and epigenetic changes, including inactivation of tumor suppressor genes. We previously cloned human G protein-coupled receptor family C type 5A (GPRC5A), whose expression is suppressed in some human lung carcinoma cells, and its mouse homolog Gprc5a.We generated Gprc5a knockout mice by homologous recombination and studied their phenotype by macroscopic observation and microscopic histologic analysis of embryos and lungs of 1- to 2-year-old mice. GPRC5A mRNA expression was analyzed by reverse transcription-polymerase chain reaction in surgical specimens of 18 human lung tumors and adjacent normal tissues and by analyzing previously published data from 186 lung tumor tissues of a variety of histologic types and 17 normal lung samples. Human embryonic kidney, human non-small-cell lung cancer, and mouse lung adenocarcinoma cells were transfected with a GPRC5A expression vector or a control vector, and colony formation in semisolid medium was assayed. Statistical tests were two-sided.Homozygous knockout mice developed many more lung tumors at 1-2 years of age (incidence: 76% adenomas and 17% adenocarcinomas) than heterozygous (11% adenomas) or wild-type (10% adenomas) mice. Human GPRC5A mRNA levels were lower in most (11 of 18 [61%]) human lung tumors than in adjacent normal tissues. The mean GPRC5A mRNA level in adenocarcinoma (n = 139), squamous cell carcinoma (n = 21), small-cell lung cancer (n = 6), and carcinoid (n = 20) tissues was 46.2% (P = .014), 7.5% (P.001), 5.3% (P.001), and 1.8% (P.001), respectively, that in normal lung tissues (n = 17) GPRC5A transfection suppressed colony formation in semisolid medium of immortalized human embryonic kidney, human non-small-cell lung cancer, and mouse lung adenocarcinoma cells by 91%, 91%, and 68%, respectively, compared with vector controls (all P.001).Gprc5a functions as a tumor suppressor in mouse lung, and human GPRC5A may share this property. The Gprc5a-deficient mouse is a novel model to study lung carcinogenesis and chemoprevention.

Recently, our laboratory demonstrated that Stat3 is required for the de novo development of chemically-induced skin tumors. We have further investigated the role of Stat3 in epithelial carcinogenesis using mice in which the expression of a constitutively active/dimerized form of Stat3 (Stat3C) is targeted to the proliferative compartment of epidermis (referred to as K5.Stat3C transgenic mice). Keratinocytes from K5.Stat3C mice showed increased survival following exposure to 7,12-dimethylbenz[a]anthracene (DMBA) and enhanced proliferation following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). In two-stage chemical carcinogenesis experiments using DMBA as the tumor initiator and TPA as the promoter, K5.Stat3C mice developed skin tumors with a shorter latency and in much greater number compared to non-transgenic littermates. Remarkably, 100% of the skin tumors that developed in K5.Stat3C transgenic mice bypassed the premalignant stage and were initially diagnosed as carcinoma in situ which rapidly progressed to squamous cell carcinoma (SCC). These tumors were highly vascularized, poorly differentiated and invasive and loss of expression of K10, filaggrin and E-cadherin was observed by 20 weeks. Finally, overexpression of Stat3C in a papilloma cell line led to enhanced cell migration and enhanced invasion through Matrigel in both the absence and presence of growth factors. In addition to its critical role in early stages of epithelial carcinogenesis, the current study reveals a novel role for Stat3 in driving malignant progression of skin tumors in vivo.

Purpose To conduct a phase III trial of adjuvant 13-cis-retinoic acid (13cRA) plus interferon alfa (IFN-α) for preventing tumor recurrence and second primary tumors (SPTs) of skin squamous cell carcinoma (SCC) among patients with aggressive skin SCC. Patients and Methods Sixty-six patients with aggressive skin SCC were randomly assigned to receive either 6 months of combined 13cRA (1 mg/kg/d orally) and IFN-α (3 × 106 U subcutaneously three times per week) or no adjuvant therapy (control group) after SCC surgery and/or radiation. Results At 21.5 months median follow-up, treatment did not improve the time to tumor recurrence and SPT versus control (hazard ratio [HR], 1.13; 95% CI, 0.53 to 2.41), time to tumor recurrence (HR, 1.08; 95% CI, 0.43 to 2.72), or time to SPT (HR, 0.89; 95% CI, 0.27 to 2.93). Adjuvant 13cRA and IFN-α was moderately tolerable; 29% of patients in the treatment arm required dose reductions for grade 3 or 4 toxicities. Conclusion Results of this phase III trial do not support 13cRA plus IFN-α for adjuvant therapy of aggressive skin SCC. With high rates of tumor recurrence and SPTs, patients with aggressive skin SCC continue to have an unmet medical need, with devastating mortality, morbidity, and financial consequences. Promising agents with preclinical and early clinical results relevant to aggressive skin SCC deserve a high priority for future clinical drug development.

Despite the use of retinoids in the clinic for many years, their mode of action in the prevention of skin cancer is still unclear. Recent microarray analyses of the chemopreventive effect of all-trans retinoic acid (ATRA), one of the primary naturally occurring biologically active retinoids, in the two-stage mouse skin chemical carcinogenesis model have provided novel insight into their action. Comparison of the gene expression profiles of control skin to skin subjected to the two-stage protocol for 3 wk, with or without ATRA, has shown that approximately half of the genes regulated by 12-o-tetradecanoylphorbol-13-acetate (TPA) are oppositely regulated when ATRA is coadministered with TPA. It was further shown the Raf/Mek/Erk branch of mitogen-activated protein (MAP) kinase pathway contains a disproportionate number of oppositely regulated genes, thereby implicating it as one of the key pathways involved in tumor promotion by TPA, that is blocked by ATRA. This result has pointed the way toward the detailed study of Raf/Mek/Erk pathway signaling in skin cancer development and its potential as a target pathway for chemoprevention by ATRA and other chemopreventive drugs.

Background Neuropathic pain is evasive to treat once developed, however evidence suggests that local administration of anesthetics near the time of injury reduces the development of neuropathic pain. As abnormal electrical signaling in the damaged nerve contributes to the initiation and maintenance of neuropathic pain, local administration of anesthetics prior to injury may reduce its development. We hypothesized that local treatment with bupivacaine prior to nerve injury in a rat model of spinal nerve ligation (SNL) would attenuate the initiation and/or maintenance of neuropathic pain behaviors. Methods On the day prior to SNL, baseline measures of pre-injury mechanical, thermal, and/or cold sensitivity were recorded in adult male Sprague–Dawley rats. Immediately prior to SNL or sham treatment, the right L5 nerve was perineurally bathed in either 0.05 mL bupivacaine (0.5 %) or sterile saline (0.9 %) for 30 min. Mechanical allodynia, thermal hyperalgesia, and/or cold allodynia were then examined at 3, 7, 10, 14 and 21 days following SNL. Results Rats exhibited both mechanical and cold allodynia, but not thermal hyperalgesia, within 3 days and up to 21 days post-SNL. No significant pain behaviors were observed in sham controls. Preemptive local bupivacaine significantly attenuated both mechanical and cold allodynia as early as 10 days following SNL compared to saline controls and were not significantly different from sham controls. Conclusions These data indicate that local treatment with bupivacaine prior to surgical manipulations that are known to cause nerve damage may protect against the maintenance of chronic neuropathic pain.

Combat injuries result in acute, severe pain. Early use of analgesia after injury is known to be beneficial. Studies on prehospital analgesia in combat are limited and no prospectively designed study has reported the use of analgesics in the prehospital and en route care setting. Our objective was to describe the current use of prehospital analgesia in the combat setting.This prospectively designed, multicenter, observational, prehospital combat study was undertaken at medical treatment facilities (MTF) in Afghanistan between October 2012 and September 2013. It formed part of a larger study aimed at describing the use of lifesaving interventions in combat. On arrival at the MTF, trained on-site investigators enrolled eligible patients and completed standardized data capture forms, which included the name, dose, and route of administration of all prehospital analgesics, and the type of provider who administered the drug. Physiological data were retrospectively ascribed as soon as practicable. The study was prospectively approved by the Brooke Army Medical Center institutional review board.Data were collected on 228 patients, with 305 analgesia administrations recorded. The predominant mechanism of injury was blast (50%), followed by penetrating (41%), and blunt (9%). The most common analgesic used was ketamine, followed by morphine. A combination of analgesics was given to 29% of patients; the most common combination was ketamine and morphine. Intravenous delivery was the most commonly used route (55%). Patients transported by the UK Medical Emergency Response Team (MERT) or U.S. Air Medical Evacuation (Dust-off) team were more likely to receive ketamine than those evacuated by U.S. Pararescue Jumpers (Pedro). Patients transported by Medical Emergency Response Team or Pedro were more likely to receive more than 1 drug. Patients who received only ketamine had a higher pulse rate (p0.005) and lower systolic blood pressure (p=0.01) than other groups, and patients that received hydromorphone had a lower respiratory rate (p=0.04).In our prospectively designed, multicenter, observational, prehospital combat study, ketamine was the most commonly used analgesic drug. The most frequently observed combination of drugs was ketamine and morphine. The intravenous route was used for 55% of drug administrations.

Retinoid X receptors (RXRs) comprise a family of nuclear retinoid activated transcription factors that are members of the steroid hormone receptor superfamily. RXRs are obligate heterodimerization partners with several other hormone receptor family members, making them critical mediators of a wide range of signaling pathways. Retinoids have been used successfully for the prevention of a number of epithelial cancers, including skin squamous cell carcinoma (SCC). The reduced expression levels of retinoid receptors including RXRalpha, the predominant RXR expressed in skin, is associated with malignancy in skin SCC. In order to study the regulation of RXRalpha in skin SCC carcinogenesis we have previously mapped the majority of the human RXRalpha gene. In the present study we have identified its first exon and promoter region. Exon 1, which contains the translation start site, is located in a highly G+C rich region of the genome at least 58 kb centromeric from exon 2. The promoter region itself is unusually G+C rich (75% G+C in 1200 bp of upstream sequence), has 17 putative SP1 transcription factor binding sites and no TATA or CAAT boxes. Transient transfection experiments with RXRalpha promoter-luciferase reporter constructs in SRB12-p9 skin SCC cells, as well as with PC3 prostate carcinoma cells, revealed that RXRalpha transcription is relatively weak compared to the positive control thymidine kinase (TK) promoter and is stimulated by treatment with all-trans retinoic acid (ATRA), the biologically active form of vitamin A. These results indicate that the RXRalpha gene is transcribed at stable levels, similar to most housekeeping genes, and its transcription is regulated by ATRA. In addition, the 5' untranslated region of RXRalpha is highly G+C rich, resulting in a potentially stable folding pattern, that would place RXRalpha amongst a group of genes that are subject to regulation at the translational level.

XAF1 (XIAP-associated factor 1) binds to XIAP and blocks its anti-apoptotic activity. It has been reported that XAF1 is mainly expressed in normal tissues but is missing or present at low levels in most cancer cell lines, which implies a tumor-suppressing function. In the present study we describe the identification of a novel splice variant of human XAF1, designated XAF1C, which contains a cryptic exon. Incorporation of this exon (exon 4b) into the mRNA introduces an in-frame stop codon, resulting in a shortened open-reading frame (ORF) of 495 nucleotides. This ORF is predicted to encode a 164 amino acid (AA) protein lacking the C-terminal domain of the previously described XAF1(A), but containing a unique 24 AA carboxy terminus. Like XAF1(A), XAF1C mRNA expression was detected in a variety of human cancer cell lines and also in normal human tissues. The ratio of XAF1(A) and XAF1C mRNA expression differs amongst the cell lines tested, suggesting differential mRNA stabilities and/or the existence of tissue- or cell type-specific splicing regulation. In transfected cells, xaf1c encodes a truncated protein of 18kDa, which is distributed primarily in the nucleus.

Here we report that epidermal keratinocytes in psoriatic lesions are characterized by activated Stat3. Transgenic mice with keratinocytes expressing a constitutively active Stat3 (K5.Stat3C mice) develop a skin phenotype either spontaneously, or in response to wounding, that closely resembles psoriasis. Keratinocytes from K5.Stat3C mice show upregulation of several molecules linked to the pathogenesis of psoriasis. In addition, the development of psoriatic lesions in K5.Stat3C mice requires cooperation between Stat3 activation in keratinocytes and activated T cells. Finally, abrogation of Stat3 function by a decoy oligonucleotide inhibits the onset and reverses established psoriatic lesions in K5.Stat3C mice. Thus, targeting Stat3 may be potentially therapeutic in the treatment of psoriasis.

Glycogen synthase kinase-3beta (GSK-3beta) activity is suppressed when it becomes phosphorylated on serine 9 by protein kinase B (Akt). To determine how GSK-3beta activity opposes Akt function we used various methods to alleviate GSK-3beta suppression in prostate carcinoma cells. In some experiments, LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (a kinase involved in activating Akt) and tumor necrosis factor-alpha (TNF-alpha) were used to activate GSK-3beta. In other experiments mutant forms of GSK-3beta, GSK-3betadelta9 (a constitutively active deletion mutant of GSK-3beta) and GSK-3betaY216F (an inactive point mutant of GSK-3beta) were used to alter GSK-3beta activity. LY294002, TNF-alpha, and overexpression of wild-type GSK-3beta or of GSK-3betadelta9, but not GSK-3betaY216F, alleviated the suppression of GSK-3beta activity in prostate carcinoma cells and enhanced the turnover of beta-catenin. Forced expression of wild-type GSK-3beta or of GSK-3betadelta9, but not GSK-3betaY216F, suppressed cell growth and showed that the phosphorylation status of GSK-3beta can affect its intracellular distribution. When transcription factors activator protein-1 and cyclic AMP-response element (CRE)-binding protein were analyzed as targets of GSK-3beta activity, overexpression of wild-type GSK-3beta suppressed AP1-mediated transcription and activated CRE-mediated transcription. Overexpression of GSK-3betadelta9 caused an (80-fold) increase in CRE-mediated transcription, which was further amplified (up to 130-fold) by combining GSK-3betadelta9 overexpression with the suppression of Jun activity. This study also demonstrated for the first time that expression of constitutively active GSK-3betadelta9 results in the phosphorylation of CRE-binding protein on serine 129 and enhancement of CRE-mediated transcription in intact cell nuclei.

PURPOSE: The purpose of this study was to test interferon alfa (IFNα), 13-cis-retinoic acid (13cRA), and cisplatin biochemotherapy in advanced squamous cell carcinoma (SCC) of the skin. PATIENTS AND METHODS: Patients with advanced skin SCC received IFNα (5 × 106 IU/m2, subcutaneous injection, three times a week), 13cRA (1 mg/kg, orally, daily), and cisplatin (20 mg/m2, intravenous injection, weekly) in a phase II trial. The growth inhibition, cell-cycle, and apoptosis activity of these agents was evaluated in two skin SCC cell lines (SRB1-m7 and SRB12-p9). RESULTS: Thirty-nine patients were enrolled. All were assessable for survival, 35 for response and toxicity (median follow-up was 38 months). The overall and complete response rates were 34% and 17%, respectively, with median durations of 9 and 35.4 months, respectively. The response rate was higher in locally advanced (67%) than metastatic (17%) disease (P = .007). Median survival was 14.6 months. One-, 2-, and 5-year survival rate estimates were 58%, 32%, and 21%, respectively. Toxicity included generally mild to moderate fatigue and mucocutaneous dryness, moderate to severe neutropenia (38%), and neutropenic fever (6%). There were no treatment-related deaths. In vitro growth inhibition and apoptosis effects of cisplatin were differential and inversely associated with those of retinoic acid and especially IFNα in two skin SCC lines. CONCLUSION: The rising incidence, morbidity, and mortality of advanced skin SCC are a major challenge for clinical oncologists. Combined 13cRA, IFNα, and cisplatin was clinically active in extensive locally advanced disease. Each agent had independent, non–cross-resistant biologic effects in vitro, which may account for the combination’s clinical activity.

Insulin-like growth factor-binding protein (IGFBP)-3 regulates apoptosis in an IGF-independent fashion and has been shown to localize to nuclei. We cloned the nuclear receptor retinoid X receptor-alpha(RXR-alpha) as an IGFBP-3 protein partner in a yeast two-hybrid screen. Multiple methodologies showed that IGFBP-3 and RXR-alpha bind each other within the nucleus. IGFBP-3-induced apoptosis was abolished in RXR-alpha-knockout cells. IGFBP-3 and RXR ligands were additive in inducing apoptosis in prostate cancer cells. IGFBP-3 enhanced RXR response element and inhibited RARE signaling. Thus, RXR-alpha-IGFBP-3 interaction leads to modulation of the transcriptional activity of RXR-alpha and is essential for mediating the effects of IGFBP-3 on apoptosis.

Managing burn injury-associated pain and wounds is a major unresolved clinical problem. Opioids, nonsteroidal antiinflammatory drugs (NSAIDs), antidepressants and anticonvulsants remain the most common forms of analgesic therapy to treat burn patients. However, prolonged treatment with these drugs leads to dose escalation and serious side effects. Additionally, severe burn wounds cause scarring and are susceptible to infection. Recent encouraging findings demonstrate that curcumin, a major bioactive component found in turmeric, is a natural pharmacotherapeutic for controlling both severe burn pain and for improved wound healing.This article covers current pr-clinical and clinical studies on the analgesic and wound healing effects. Particular emphasis has been placed on studies aimed at developing improved curcumin delivery vehicles that increase its bioavailability. Based on the available evidence, a hypothesis is proposed that the dual beneficial effects of curcumin, analgesia and enhanced wound healing are mediated through common anti-inflammatory mechanisms.Emerging studies have demonstrated that curcumin is a promising investigational drug to treat both pain and wounds. The adequate control of severe burn pain, particularly over the long courses required for healing, as well improvements in burn wound healing are unmet clinical needs.

Opioid-related side effects are problematic for burn patients. Dual mechanism therapeutics targeting opioid and non-opioid mechanisms may have reduced side effects with similar analgesic efficacy. Tramadol combines mu opioid receptor agonism with norepinephrine reuptake inhibition and has been effective in treating some types of pain. The effectiveness of tramadol in treating pain associated with burns is unclear. We hypothesized that tramadol is effective in reducing thermal injury-evoked pain behaviors in a rat model. Rats were anesthetized and a 100°C metal probe was placed on the hindpaw for 30 s to induce a full thickness thermal injury. A subset of rats was perfusion fixed and hindpaw tissue and spinal cord collected for anatomical analysis. Rats received morphine (5 mg/kg; i.p.), tramadol (10-30 mg/kg; i.p.) or vehicle and latency to paw withdrawal from a noxious thermal or non-noxious mechanical stimulus was recorded every 10 min over 70 min and again at 2 h. We report that pain behaviors developed within 48 h and peaked at 1 week; paralleled by enhanced expression of pronociceptive neuropeptides in the spinal cord. Morphine and tramadol significantly attenuated hyperalgesia and allodynia, while not significantly altering motor coordination/sedation. These data indicate dual mechanism therapeutics may be effective for treating pain associated with burns.

The F9 murine embryonal carcinoma (EC) cell line, a well established model system for the study of retinoic acid (RA)-induced differentiation, differentiates into cells resembling three types of extra-embryonic endoderm (primitive, parietal and visceral), depending on the culture conditions and RA concentration used. A number of previously identified genes are differentially expressed during this process and serve as markers for the different endodermal cell types. Differentiation is also accompanied by a decreased rate of proliferation and an apoptotic response. Using homologous recombination, we have disrupted both alleles of the retinoid X receptor (RXR) alpha gene in F9 cells to investigate its role in mediating these responses. The loss of RXRalpha expression impaired the morphological differentiation of F9 EC cells into primitive and parietal endoderm, but has little effect on visceral endodermal differentiation. Concomitantly the inducibility of most primitive and parietal endoderm differentiation-specific genes was impaired, while several genes upregulated during visceral endodermal differentiation were induced normally. We also demonstrate that RXRalpha is required for both the anti-proliferative and apoptotic responses in RA-treated F9 cells. Additionally, we provide further evidence that retinoic acid receptor (RAR)-RXR heterodimers are the functional units transducing the effects of retinoids in F9 cells.

The purpose of the current study was to determine whether a tropical ginger derived compound 1’-acetoxychavicol acetate (ACA), suppresses skin tumor promotion in K5.Stat3C mice. In a two-week study in which wild-type (WT) and K5.Stat3C mice were co-treated with either vehicle, ACA, galanga extract, or fluocinolone acetonide (FA) and tetradecanoyl phorbol acetate (TPA), only the galanga extract and FA suppressed TPA-induced skin hyperproliferation and wet weight. None of these agents were effective at suppressing p-Tyr705Stat3 expression. However, ACA and FA showed promising inhibitory effects against skin tumorigenesis in K5.Stat3C mice. ACA also suppressed phospho-p65 NF-κB activation, suggesting a potential mechanism for its action.

Analysis of high-dimensional micro array expression data is based mostly on the statistical approaches that are indispensable for the study of biological systems. To aid the analysis and exploration of such data, the process of analyzing such data is often enhanced with visual, data mining and other computational techniques. We utilize a set of tools for the visual analysis of data aimed at generating the hypotheses. We show the usability of classic and novel multi-dimensional visualization tools in life sciences. Additionally, we survey and show several multidimensional visualization tools applied to the process of data exploration using a urothelial cell carcinoma of the bladder time course. These tools have the potential of uncovering non-trivial relationships and structures in the data.

Background Stat3 is a cytokine- and growth factor-inducible transcription factor that regulates cell motility, migration, and invasion under normal and pathological situations, making it a promising target for cancer therapeutics. The hepatocyte growth factor (HGF)/c-met receptor tyrosine kinase signaling pathway is responsible for stimulation of cell motility and invasion, and Stat3 is responsible for at least part of the c-met signal. Methods We have stably transfected a human squamous cell carcinoma (SCC) cell line (SRB12-p9) to force the expression of a dominant negative form of Stat3 (S3DN), which we have previously shown to suppress Stat3 activity. The in vitro and in vivo malignant behavior of the S3DN cells was compared to parental and vector transfected controls. Results Suppression of Stat3 activity impaired the ability of the S3DN cells to scatter upon stimulation with HGF (c-met ligand), enhanced their adhesion, and diminished their capacity to invade in vitro and in vivo. Surprisingly, S3DN cells also showed suppressed HGF-induced activation of c-met, and had nearly undetectable basal c-met activity, as revealed by a phospho-specific c-met antibody. In addition, we showed that there is a strong membrane specific localization of phospho-Stat3 in the wild type (WT) and vector transfected control (NEO4) SRB12-p9 cells, which is lost in the S3DN cells. Finally, co-immunoprecipitation experiments revealed that S3DN interfered with Stat3/c-met interaction. Conclusion These studies are the first confirm that interference with the HGF/c-met/Stat3 signaling pathway can block tumor cell invasion in an in vivo model. We also provide novel evidence for a possible positive feedback loop whereby Stat3 can activate c-met, and we correlate membrane localization of phospho-Stat3 with invasion in vivo.

Squamous cell carcinoma (SCCa) has increased from 4% to 10% over 4 decades, stimulating interest in developing novel agents that slow sun-damaged skin progression. This is the first study evaluating the naturally occurring bioactive food compound curcumin on skin cancer xenografts. Low bioavailability of curcumin has slowed its transition to clinical trials. It is hypothesized that curcumin has growth-inhibitory effects through the TOR pathway and chemopreventive potential in skin SCCa where local application could bypass bioavailability problems.A randomized experimental animal and laboratory study.Louisiana State University Health Sciences Center, Shreveport, Louisiana.SCID mice were pretreated with 0, 5, or 15 mg of curcumin (n = 8 per group), 3 days prior to injecting 10⁶ SRB12-p9 skin SCCa cells in each flank, and were gavaged daily thereafter. Tumor volumes were measured and tumors were harvested on day 24 when mice were sacrificed. Immunohistochemical analysis of pS6 expression (n = 3 per group) and tumor volumes in the 3 groups were compared using 1-way analysis of variance and pairwise comparisons were determined with the Tukey t test if overall comparisons were significant.Tumor volume increased 2.3 times faster in control mice compared with the group receiving 15 mg of curcumin (P = .0003). A significant difference in average tumor volumes was seen (P = .0012), especially with treatment of 15 mg of curcumin compared with control P = .0003). Curcumin inhibited S6 phosphorylation (P = .0027), suggest-ing inhibition of the MTOR pathway.Curcumin appears to inhibit skin SCCa growth and blocks tumor progression by inhibiting pS6 even when gavage is used to deliver curcumin, indicating even more significant effects in future experiments with local application.

Integrin α6β4 is the receptor for the laminin family of extracellular matrix proteins and is widely expressed in most epithelial tissues and Schwann cells. The expression of this integrin is up-regulated in most epithelial tumors, suggesting the role of α6β4 in their progression. The tumor microenvironment is also known to enhance the signaling competence of α6β4 through functional and physical interactions with other receptors. In this review, we discuss the biological mechanisms by which integrin α6β4 promotes carcinoma cell motility and invasion that leads to mammary tumor progression.

Ultraviolet (UV) radiation-induced immunosuppression has been implicated in the development of skin cancers. As oral administration of green tea polyphenols (GTPs) in drinking water prevents photocarcinogenesis in mice, we studied whether administration of GTPs in drinking water of mice (0.1-0.5%, w/v) prevents UV-induced immunosuppression, and determined the possible mechanism of action of GTPs. We observed that GTPs (0.2 and 0.5%, w/v) prevented UV-induced suppression of contact hypersensitivity (CHS) response to a contact sensitizer in local (58-62%, p