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NIAMS • 1K08 AR054388
Karen Taraska Hastings, MD, PhD
Melanoma is the most lethal form of skin cancer. Immunotherapy holds great promise in the treatment of metastatic melanoma because melanoma antigens have been identified and patients generate T and B cell responses specific for these antigens which in some cases lead to spontaneous remission. Effective immunotherapy requires presentation of tumor antigens in the context of MHC class II for the activation of CD4+ T lymphocytes to generate and sustain the anti-tumor immune response. Additionally, since many melanoma antigens represent self-antigens present in benign melanocytes, a productive anti-tumor response requires the avoidance of tolerance mechanisms which suppress the immune response to self-antigens. These studies will identify key features of antigen processing required to generate an immune response to melanoma and may aid in the understanding of mechanisms that lead to immune evasion. The long-term goal of these studies is to aid the development of effective immunotherapy for melanoma.
1. Evaluate the role of GILT in generating tolerance to melanocyte differentiation antigen TRP-1
2. Determine the role of GILT in generation of immune responses to melanocyte differentiation antigens
3. Determine whether GILT expression correlates with disease progression using immunohistochemistry.
Since the majority of melanoma antigens represent self-antigens, avoiding central and peripheral tolerance is essential for productive anti-tumor immunity. Controlled expression of GILT in thymic and peripheral antigen presenting cells may have the potential to regulate the balance between T cell activation and tolerance. Another important objective of this study is to evaluate the expression of GILT in comparison to class II in human melanoma tissue samples and determine whether GILT expression correlates with presence of tumor infiltrating lymphocytes and melanoma severity at diagnosis. Altering GILT expression and MHC class II antigen processing may be important mechanisms by which melanomas evade immune-mediated destruction.
These studies will identify key features of antigen processing required to generate an immune response to melanoma and may aid in the understanding of mechanisms that lead to immune evasion. The long-term goal of these studies is to aid the development of effective immunotherapy for melanoma.
We propose to address the role of MHC class II antigen processing components, in particular gamma-interferon inducible lysosomal thiol reductase (GILT), in the immune recognition of melanoma. Melanoma antigens including tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2 and gp100 are likely to be substrates for lysosomal reduction by GILT, because these melanosomal membrane proteins are present in the MHC class II loading compartment and contain internal disulfide bonds. We have demonstrated that GILT is essential for the MHC class II processing of TRP-1 in vitro. In this proposal, we plan to explore the role of GILT in the development of tolerance to TRP-1 using a TRP-1-specific T cell receptor transgenic mouse model. We next plan to determine the role of GILT in the overall immune response to tyrosinase, TRP-1, TRP-2 and gp100 and to evaluate the role of GILT in protection from melanoma challenge by comparing in vivo immune responses in GILT-deficient compared to wild-type mouse strains. To explore the relevance in human disease, we will determine the expression of GILT in human melanoma.
NCI • 5K07 CA132959
Sally Dickinson, PhD
Skin cancer is one of the most prevalent diseases of the modern world. This year, approximately 1.2 million new cases of skin cancer will be diagnosed in the United States, which will account for roughly 40% of all new cancer cases. The majority of these skin cancer cases will be classified as non-melanoma skin cancer (NMSC). NMSCs occur primarily on sun-exposed areas of the body and have been attributed to cumulative cellular changes brought about by ultraviolet (UV) light radiation. Sulforaphane (SF) is a naturally-occurring isothiocyanate present in cruciferous vegetables and is an established chemopreventive agent in several cancer models. Both earlier work and our own published data indicate that SF is effective at preventing UVB-induced NMSC in mice. However, the mechanism of this inhibition is uncertain. The chemopreventive effect of SF is typically thought to stem from its ability to activate the transcription factor Nf-E2 related factor-2 (Nrf2). Recent evidence suggests that SF may also selectively inhibit the transactivation potential of the tumor promoting transcription factor activator protein-1 (AP-1) after UVB. We propose that inhibition of UVB-induced NMSC by SF requires the coordinated regulation of both Nrf2 and AP-1. Additionally, clinical trials are required to study the safety and efficacy of purified SF when applied to the skin. Understanding the relative effects of SF on Nrf2 activation and AP-1 inhibition at the molecular, cellular and organismal levels will aid in our ability to safely move SF into long-term clinical trials for NMSC chemoprevention
1. Test whether the inhibitory effect of SF on AP-1 transactivation and skin carcinogenesis is due to chemical interactions with the DNA binding domain of cFos and/or cJun.
2. Test the influence of SF-induced Nrf2 on protection against acute UVB-induced mouse skin damage and chronic UVB-induced NMSC.
3. Test the safety and efficacy of topical purified SF in humans (Phase I/IIa clinical trial).
Although there is heightened awareness about the need for limited sun exposure and use of sunscreens, this has not significantly impacted NMSC incidence. Therefore, we must search for better methods to prevent NMSC, in formats which are affordable and manageable for public use. The natural product sulforaphane may be an excellent candidate for use in the prevention of NMSC caused by UV exposure, since it targets at least two pathways involved in carcinogenesis: Nrf2 and AP-1. This work will help to shed light on the relative importance of each of these pathways in relation to the chemopreventive action of SF against NMSC.
New topical formulations of SF will be tested for possible translation to human subjects. In addition, a new inducible, tissue-specific transgenic mouse will be generated for this project to determine whether mutation of the DNA binding domain of cFos will block the effectiveness of SF chemoprevention in the skin.
We have found that in addition to activation of Nrf2, SF inhibits AP-1 transactivation after UVB. We hypothesize that this inhibition is due to the interaction of SF with redox-sensitive cysteines present in the DNA binding domains of AP-1 family members such as cFos and cJun. Mutation of this cysteine should result in constitutive DNA binding and AP-1 activation. We will test this hypothesis in human keratinocytes using ChIP assays to assess whether SF or other cysteine oxidizing agents can block AP-1 DNA binding after UVB. We will also create transgenic mice which express an epidermal, inducible form of cFos in which the redox-sensitive cysteine in the DNA-binding domain is mutated. This mutation should result in higher rates of chronic UVB-induced carcinogenesis, and less response to SF pretreatment than is found in wildtype littermates.
In addition, we will use Nrf2 knockout mice to test whether SF can still play a role in chemoprevention in the absence of this target. SF pretreatment should decrease the oxidative damage incurred by acute UVB exposure in wildtype mice but not in Nrf2 knockout mice. However, SF should inhibit AP-1-linked biomarkers in both genotypes. Chronic exposure to UVB in Nrf2 knockout and wildtype mice will also be performed. Nrf2 knockout mice should still respond to SF pretreatment with reduced tumor burden and incidence, but this reduction will be much more significant in the wildtype animals due to the ability of SF to affect both AP-1 and Nrf2.
Finally, we plan to perform an acute Phase 0 trial testing SF on the skin of human volunteers to determine whether biomarkers for proliferation, apoptosis, Nrf2 and AP-1 activity can be affected in vivo after UVB exposure. Pretreatment with SF should reduce redness associated with UVB treatment, and should increase the expression of cytoprotective and antioxidant enzymes in the skin. SF may therefore be a good candidate for addition to sunscreens and lotions to help prevent UV-induced skin damage.
M Peter Lance, MD
Colorectal cancer (CRC) is the second most common cause of cancer death in the United States. It was estimated that in 2005 there would be 145,290 new cases and 56,290 deaths from the disease. CRCs, like other neoplasms, develop from normal epithelium by acquiring sequential malfunction of multiple genes before progression to invasive malignancy occurs. Adenomatous polyps of the colon and rectum, collectively termed colorectal adenomas (CRAs), are benign neoplasms and precursors, through the adenoma-carcinoma sequence, of CRC . An additional variety of colorectal polyp, the ‘serrated adenoma’, has recently been recognized as having malignant potential. Development of an early CRA from hyper-proliferative epithelium marks the initiation of colorectal neoplasia. As already described, the 10% or fewer of CRAs at greatest risk for progressing to CRC are termed adenomas with advanced pathology, abbreviated as advanced adenomas or, in this proposal, advanced CRAs. Advanced CRAs and CRCs together comprise advanced colorectal neoplasms, which are the primary targets of colorectal screening by fecal occult blood testing, sigmoidoscopy or colonoscopy. Most CRAs can be completely removed by the endoscopic technique of polypectomy. However, new, so called metachronous CRAs develop at a rate of 10-15% per year in subjects already diagnosed with CRAs, who, therefore, should undergo periodic surveillance colonoscopy for diagnosis and removal of metachronous CRAs. Chemoprevention is an additional option to surveillance colonoscopy, particularly for subjects with a prior history of advanced colorectal neoplasms. The FDA Gastrointestinal Drugs Advisory Committee has made recommendations for CRC chemoprevention trials, including that: (1) CRAs may serve as meaningful endpoints in cancer prevention trials. (2) Trials should include supportive data on increases in the number of CRA-free patients. (3) Trials should be well controlled, ensure adequate compliance, and last 3 to 6 years.
The primary objective of the SELECT Trial is to assess the effect of L-selenomethionine and vitamin E alone and in combination on prostate cancer incidence. The trial is being conducted in 35,584 men, who will take trial supplements for 7 to 12 years. Completion of the trial is anticipated in 2013. Planned secondary objectives of SELECT include assessment of the effect of selenium and vitamin E on colorectal cancer incidence and colorectal cancer-free survival. Significance
Randomized trials comparing antioxidant supplements with placebo for prevention of colorectal and other gastrointestinal cancers were reviewed recently using the Cochrane methodology. With the exception of selenium, antioxidants tended to increase rather than decrease overall mortality. Selenium, however, seemed to show significant beneficial effect in four trials, although methodology was considered sub-optimal in these trials. The authors concluded that “The potential preventive effect of selenium should be studied in adequate randomised trials”. Our Selenium Clinical Trial, a CRA recurrence trial, is such a trial.
A unique aspect of the proposed Colorectal SELECT Ancillary Study is the opportunity to measure the effects of SELECT interventions on advanced CRAs and CRC as well as on CRA initiation.
In the current proposal, we will investigate effect modification of concomitant aspirin use on the efficacy of the selenium and vitamin E interventions for chemoprevention of colorectal neoplasia. As a secondary objective we will investigate effect modification of the selenium and vitamin E interventions by BMI.
SELECT participants diagnosed with CRAs will be identified through a questionnaire that will be administered every six months to all participants at their home Clinical Center. The questionnaire will ask the participant whether he has undergone lower endoscopy (sigmoidoscopy or colonoscopy) since this question was last asked. Those who have undergone lower endoscopy will be asked for: 1) consent to release their names to Colorectal SELECT staff at the Arizona Cancer Center and 2) contact information of the physicians who performed the endoscopies. Study staff at the Arizona Cancer Center will obtain medical records of the endoscopies, and histopathology reports and sections or blocks of all polyps removed at these procedures. We estimate that we will need to access ~14,500 of the 35,584 SELECT participants in order to identify sufficient participants with CRAs for adequate statistical power. We anticipate needing to enroll up to 75 of the SELECT Study Centers for this purpose. We estimate that 55% of SELECT participants will undergo lower endoscopy while in the trial, yielding lower endoscopies in 8,000 of ~14,500, i.e., 4,000 randomized to selenium (or vitamin E) and 4,000 to placebo. We assume that 27% of participants undergoing lower endoscopy will have one or more CRAs, yielding CRAs in 2,160 of 8,000 participants, i.e., 1,080 on selenium (or vitamin E) and 1,080 on placebo. With this design, we will have 87% power to detect a 20% reduction in the occurrence of CRAs in the selenium (or vitamin E) group compared to the placebo group. CRC cases will be ascertained from the entire 35,584 SELECT population by methods already in place for identification of all cases of non-prostate cancers. Assuming a CRC incidence rate of approximately 2 cases/1000 person-years, we estimate conservatively that we will have 90% power to detect a 33% reduction in CRC incidence in participants receiving selenium (or vitamin E) compared to those on placebo.
NCI • R01 CA149417-01A1
Cynthia Thomson, PhD, RD, CSO
The treatment of breast cancer has progressed rapidly in the past 20-30 years, particularly for women diagnosed with estrogen responsive disease. Yet, women diagnosed with breast cancer remain at an increased risk for recurrent cancer for the remainder of their lives. TAM has served as the primary therapeutic agent for the treatment of estrogen receptor positive disease for the past decade and TAM is also a common drug therapy used in the primary prevention setting by women at increased genetic risk for disease. While the therapeutic use of TAM has been clearly demonstrated, there remain concerns regarding drug resistance as well as toxicity. Elevated circulating estradiol, lack of hot flash response and lower serum endoxifen levels have all been associated with reduced clinical response to TAM and endometrial toxicity remains a primary concern11. Strategies to enhance the efficacy of TAM while at the same time targeting a reduction in toxicity are needed. Here we propose a novel approach in addressing these clinical concerns using a bioactive food-derived compound in combination with TAM.
To test our primary hypothesis, we propose a randomized, double-blind, placebo-controlled trial among 170 premenopausal treated with TAM in which women are randomized to receive 150 mg oral DIM or Placebo for a period of 18 months.
1. Assess change in breast density from baseline to 18 months post-intervention using mammogram-based breast density measures as well as a novel, quantitative fat-water ratio breast magnetic resonance imaging (FWR-MRI).
2. Evaluate the effect of 150 mg daily DIM on serum steroid hormones (estrogen, sex hormone binding globulin (SHBG)) and urinary 2-hydroxyestrone:16α-hydroxyestrone (2OHE1:16αOHE1) ratio as well as serum TAM metabolites (endoxifen) in premenopausal women treated with TAM.
3. Evaluate TAM-associated endometrial toxicity using self-reported vaginal bleeding and vaginal ultrasound in women receiving DIM (150 mg DIM daily for 18 months) in combination with TAM.
TAM is commonly prescribed for breast cancer treatment in premenopausal women. Direct demonstration of a greater risk modifying activity and/or reduced TAM toxicity TAM + DIM versus TAM alone using pharmacologic dosing of the bioavailable BioResponse™ DIM would substantiate the clinical potential of DIM to improve health outcomes in this at-risk group.
Preliminary evidence supports an enhancing effect of cruciferous for the prevention of breast cancer recurrences in women on TAM. The specific mechanisms for this are not well characterized. We speculate that, based on strong pre-clinical evidence, the effects of diindolylmethane (DIM), a bioactive of cruciferous vegetables with demonstrated chemopreventive properties in animal models, on select breast cancer risk factors. Specifically, we seek to demonstrate that exposure to TAM + DIM (vs TAM alone) will result in greater reduction in breast density, a reduction in estrogen as well as greater C-2 hydroxylation and a potential elevation in the levels of endoxifen as the active metabolite of TAM. We also plan to evaluate the potential of DIM to reduce the endometrial toxicity associated with TAM use. Research to identify evidence-based diet-related strategies to significantly improve the efficacy or reduce toxicity of existing pharmaceuticals is an understudied area of translational research
This is a phase II trial of 150 mg DIM given daily for 18 months in women taking TAM to test the primary study hypothesis that DIM given orally in capsular form will be associated with a greater reduction in breast density as well as modulation of circulating hormone levels to reduce estrogen exposure. Further, these proposed improvements in breast cancer risk factors with TAM+DIM (as compared to TAM alone) will be accompanied by a reduction in TAM toxicity related to endometrial health.