Janine G. Einspahr

Pronounced reduction in adenoma recurrence associated with aspirin use and a polymorphism in the ornithine decarboxylase gene

Most sporadic colon adenomas acquire mutations in the adenomatous polyposis coli gene (APC) and show defects in APC-dependent signaling. APC influences the expression of several genes, including the c-myc oncogene and its antagonist Mad1. Ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is a transcriptional target of c-myc and a modifier of APC-dependent tumorigenesis. A single-nucleotide polymorphism exists in intron 1 of the human ODC gene, which lies between two myc-binding domains. This region is known to affect ODC transcription, but no data exist on the relationship of this polymorphism to risk of colorectal neoplasia in humans. We show that individuals homozygous for the minor ODC A-allele who reported using aspirin are ≈0.10 times as likely to have an adenoma recurrence as non-aspirin users homozygous for the major G-allele. Mad1 selectively suppressed the activity of the ODC promoter containing the A-allele, but not the G-allele, in a human colon cancer-derived cell line (HT29). Aspirin (≥10 μM) did not affect ODC allele-specific promoter activity but did activate polyamine catabolism and lower polyamine content in HT29 cells. We propose that the ODC polymorphism and aspirin act independently to reduce the risk of adenoma recurrence by suppressing synthesis and activating catabolism, respectively, of colonic mucosal polyamines. These findings confirm the hypothesis that the ODC polymorphism is a genetic marker for colon cancer risk, and support the use of ODC inhibitors and aspirin, or other nonsteroidal antiinflammatory drugs (NSAIDs), in combination as a strategy for colon cancer prevention.

Increased expression of thioredoxin-1 in human colorectal cancer is associated with decreased patient survival

Thioredoxin-1 is a redox protein that, when overexpressed, causes increased cancer-cell growth and inhibited apoptosis. Thioredoxin-1 expression has been reported to be increased in several human primary tumors, but its relationship to tumor progression and patient survival has not been established. We studied the expression of thioredoxin-1 as measured with immunohistochemical staining in paraffin-embedded human normal colonic mucosa, adenomatous polyps, and primary and metastatic colorectal cancer. Thioredoxin-1 expression was not increased in 12 colorectal adenomatous polyps, compared with 8 samples of normal colonic mucosa, but was significantly increased in 12 primary colorectal cancers (P <.01). Thioredoxin-1 expression was not significantly different in primary lymph-node metastases and the primary colorectal cancer. Using colorectal cancer samples from 37 subjects for whom survival data was available, we found that thioredoxin-1 expression increased with Dukes stage, although the association was not statistically significant (P =.077). We noted a significant association between thioredoxin-1 expression and patient survival (P =.004); higher score was associated with decreased survival. When adjusted for Dukes stage, thioredoxin-1 expression showed a statistically significant association with survival (P =.012). The work shows that increased thioredoxin-1 expression is a relatively late event in colorectal carcinogenesis and provides evidence in a small group of subjects with colorectal cancer of Dukes stages A through D that thioredoxin-1 expression may be an independent marker of patient prognosis.

Analysis of the p53 gene in human precancerous actinic keratosis lesions and squamous cell cancers

A biomarker of skin cancer would be beneficial in evaluating the efficacy of potential cancer chemoprevention agents. To this end, we investigated the tumor suppressor gene p53 in precancerous actinic keratosis lesions (AK) and malignant squamous cell carcinomas (SCCs) using polymerase chain reaction and single-strand conformation polymorphism analysis (PCR-SSCP) techniques. In addition, p53 protein expression was evaluated using immunohistochemistical analysis with the PAB 1801 monoclonal antibody. Nine out of 13 (69%) SCCs and 8 of 15 (53%) AKs were positive for p53 mutations. In contrast, normal skin samples were negative for p53 mutations. Sequence analysis of AKs and SCCs showed primarily C to T transition mutations. Nuclear immunochemical staining for p53 was observed in 12/15 (80%) AK and 12/13 (92%) SSCs. These results suggest that p53 mutations may be involved in the malignant conversion of AKs to SCCs and that p53 may be useful as a biomarker to study the potential modulatory effects of cancer chemopreventive agents against skin cancer.

Chemoprevention of human skin cancer.

The incidence of skin cancer has been rising in recent years with significant effects on public health. Primary prevention has proven inadequate in impacting the incidence of skin cancer, thus stimulating the development of chemopreventive strategies. The majority of skin cancer chemoprevention studies focus on occurrence of new nonmelanoma skin cancers (NMSC) in individuals with a previous NMSC, or on reduction in the number of premalignant skin lesions such as actinic keratoses (AK). Dysplastic nevi, a likely precursor of melanoma, are also potential targets for chemoprevention strategies. Premalignant lesions are especially attractive as endpoints since they are more common than frank cancer, resulting in reduced sample size, length, and cost of clinical trials. Development of new agents that affect the pathogenesis of skin cancer will be discussed, from elucidation of molecular targets to implementation of trials designed to determine the effects of chemopreventive interventions on human skin cancer.

Skin Cancer Chemoprevention: Strategies to Save Our Skin

There are over 1 million cases of skin cancer diagnosed yearly in the United States. The majority of these are nonmelanoma (NMSCs) and are associated with chronic exposure to ultraviolet light (UV). Actinic keratosis (AK) has been identified as a precursor for SCC, but not for BCC. AKs are far more common than SCC, making them excellent targets for chemoprevention. Cancer chemoprevention can prevent or delay the occurrence of cancer in high-risk populations using dietary or chemical interventions. We have developed strategies that have rational mechanisms of action and demonstrate activity in preclinical models of skin cancer. Promising agents proceed to phase I-III trials in subjects at high risk of skin cancer. UV light induces molecular signaling pathways and results in specific genetic alterations (i.e., mutation of p53) that are likely critical to skin cancer development. UVB-induced changes serve as a basis for the development of novel agents. Targets include inhibition of polyamine or prostaglandin synthesis, specific retinoid receptors, and components of the Ras and MAP kinase signaling pathways. Agents under study include: epigallocatechin gallate (EGCG), a green tea catechin with antioxidant and sunscreen activity, as well as UVB signal transduction blocking activity; perillyl alcohol, a monoterpene derived from citrus peel that inhibits Ras farnesylation; difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase and polyamines; retinoids that target retinoid X receptors and AP-1 activity; and nonsteroidal anti-inflammatory agents that inhibit cylooxygenase and prostaglandin synthesis. We performed a series of Phase I-II trials in subjects with multiple AK. For example, a phase II randomized trial of topical DFMO reduced AK number, suppressed polyamines, and reduced p53 protein. Our goal is to develop agents for use in combination and/or incorporation into sunscreens to improve chemoprevention efficacy and reduce skin cancer incidence.

Functional protein pathway activation mapping of the progression of normal skin to squamous cell carcinoma.

Reverse phase protein microarray analysis was used to identify cell signaling derangements in squamous cell carcinoma (SCC) compared with actinic keratosis (AK) and upper inner arm (UIA). We analyzed two independent tissue sets with isolation and enrichment of epithelial cells by laser capture microdissection. Set 1 served as a pilot and a means to identify protein pathway activation alterations that could be further validated in a second independent set. Set 1 was comprised of 4 AK, 13 SCC, and 20 UIA. Set 2 included 15 AK, 9 SCCs, and 20 UIAs. Activation of 51 signaling proteins, known to be involved in tumorigenesis, were assessed for set 1 and showed that the MEK–ERK [mitogen-activated protein (MAP)/extracellular signal-regulated (ERK; MEK)] pathway was activated in SCC compared with AK and UIA, and that epidermal growth factor receptor (EGFR) and mTOR pathways were aberrantly activated in SCC. Unsupervised two-way hierarchical clustering revealed that AK and UIA shared a common signaling network activation architecture while SCC was dramatically different. Statistical analysis found that prosurvival signaling through phosphorylation of ASK and 4EBP1 as well as increased Bax and Bak expression was higher in AK compared with UIA. We expanded pathway network activation mapping in set 2 to 101 key signaling proteins, which corroborated activation of MEK–ERK, EGFR, and mTOR pathways through discovery of a number of upstream and downstream signaling molecules within these pathways to conclude that SCC is indeed a pathway activation–driven disease. Pathway activation mapping of SCC compared with AK revealed several interconnected networks that could be targeted with drug therapy for potential chemoprevention and therapeutic applications. Cancer Prev Res; 5(3); 403–13. ©2012 AACR.

Expression of vascular endothelial growth factor in early cutaneous melanocytic lesion progression.

A considerable body of evidence supports the concept that a significant number of cutaneous malignant melanomas progress through a precursor lesion or dysplastic melanocytic nevi (DN). Tumor angiogenesis likely plays a critical role in early development of melanoma, and intermediate biomarkers of angiogenesis could be useful as chemoprevention and prognostic markers.

Associations of Ki-ras Proto-oncogene Mutation and p53 Gene Overexpression in Sporadic Colorectal Adenomas with Demographic and Clinicopathologic Characteristics

In colorectal tumorigenesis, Ki-ras proto-oncogene mutation often occurs early in the adenoma-adenocarcinoma sequence, whereas mutation of the p53 gene is associated with late progression to carcinoma. We evaluated the relationship of demographic and clinicopathologic characteristics to Ki-ras mutation and p53 gene product overexpression in 1,093 baseline sporadic colorectal adenomas from 926 individuals enrolled in a phase III recurrence prevention trial. Ki-ras mutation was found in 14.7% of individuals and p53 overexpression was found in 7.0% of those tested. Multivariate analysis found older age, rectal location, and villous histology to be independently associated with Ki-ras mutation. Individuals with an advanced adenoma (≥1 cm or high-grade dysplasia or villous histology) had a 4-fold higher likelihood of Ki-ras mutation [odds ratios (OR), 3.96; 95% confidence intervals (CI), 2.54-6.18]. Ki-ras mutations in codon 12 and of the G-to-A transition type were more frequent in older individuals, whereas G-to-T transversion was more frequent in rectal adenomas than in the colon. Multivariate analysis showed that previous history of a polyp (P = 0.03) was inversely associated with p53 overexpression. Large adenoma size (≥1 cm), high-grade dysplasia, and villous histology were independently associated with p53 overexpression, with the strongest association for advanced adenomas (OR, 7.20; 95% CI, 3.01-17.22). Individuals with a Ki-ras mutated adenoma were more likely to overexpress p53 (OR, 2.46; 95% CI, 1.36-4.46), and 94.8% of adenomas with both alterations were classified as advanced (P ≤ 0.0001). Our large cross-sectional study supports the role of both Ki-ras and p53 in the progression of adenomas and shows that their molecular pathogenesis differs by anatomic location, age, and mucosal predisposition as evidenced by previous history of a polyp. (Cancer Epidemiol Biomarkers Prev 2006;15(8):1443–50)

Safety and Efficacy of Dose-Intensive Oral Vitamin A in Subjects with Sun-Damaged Skin

Purpose: Previously, we reported the results of a Phase III, placebo-controlled trial in 2,297 randomized participants with moderately severe actinic keratoses wherein 25,000 IU/day vitamin A caused a 32% risk reduction in squamous cell skin cancers. We hypothesized that dose escalation of vitamin A to 50,000 or 75,000 IU/day would be both safe and more efficacious in skin cancer chemoprevention. Experimental Design: One hundred and twenty-nine participants with severely sun-damaged skin on their lateral forearms were randomized to receive placebo or 25,000, 50,000, or 75,000 IU/day vitamin A for 12 months. The primary study end points were the clinical and laboratory safety of vitamin A, and the secondary end points included quantitative, karyometric image analysis and assessment of retinoid and rexinoid receptors in sun-damaged skin. Results: There were no significant differences in expected clinical and laboratory toxicities between the groups of participants randomized to placebo, 25,000 IU/day, 50,000 IU/day, and 75,000 IU/day. Karyometric features were computed from the basal cell layer of skin biopsies, and a total of 22,600 nuclei from 113 participants were examined, showing statistically significant, dose-response effects for vitamin A at the 25,000 and 50,000 IU/day doses. These karyometric changes correlated with increases in retinoic acid receptor α, retinoic acid receptor β, and retinoid X receptor α at the 50,000 IU/day vitamin A dose. Conclusions: The vitamin A doses of 50,000 and 75,000 IU/day for 1 year proved safe and equally more efficacious than the 25,000 IU/day dose and can be recommended for future skin cancer chemoprevention studies.

Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples

Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.