Molecular Characterization of Preneoplastic Lesions Provides Insight on the Development of Renal Tumors

Abstract

Kidneys are the second most frequent site for chemically induced cancers in rats. However, there is still limited information on direct effects of carcinogens on pathways involved in the development of kidney tumors. Since transformed tumor cells have different characteristics than their cell of origin, it was hypothesized that healthy tissue and progressing stages of preneoplastic lesions are differentially influenced by chemical carcinogens. To elucidate this question, TSC2؊/؊ Eker rats were gavaged with genotoxic aristolochic acid or nongenotoxic ochratoxin A for 3 and 6 months, respectively. Histopathology and cell proliferation analysis demonstrated a compound-and sex-specific onset of preneoplastic lesions. In contrast, comparable gene expression profiles of lasermicrodissected preneoplastic lesions from carcinogen-treated and control rats, including reduced expression of genes involved in carcinogen uptake and metabolism, point to a compound-independent lesion progression. Gene expression profiles and additional immunostaining suggested that clonal expansion of renal lesions appears primarily driven by disturbed mammalian target of rapamycin complex 1 and mammalian target of rapamycin complex 2 pathway regulation. Finally, prolonged carcinogen exposure resulted in only marginal gene expression changes in tubules with normal morphology, indicating that some tubules may have adapted to the treatment. Taken together, these findings indicate that the final outcome of in vivo carcinogenicity studies is primarily determined by time-restricted initial events, while lesion progression may be a compound-independent process, involving deregulated mTOR signaling in the Eker rat model.

Time-matched analysis of DNA adduct formation and early gene expression as predictive tool for renal carcinogenesis in methylazoxymethanol acetate treated Eker rats

“…Similarly, continuous MAMAc exposure over 3 and 6 months (Figure 5b, c) had no impact on cell proliferation when analyzed by BrdU staining. Both techniques have been successfully applied for the detection of cell proliferating differences in carcinogen or vehicle treated Eker rats (Stemmer et al 2009), indicating adequate sensitivity of both staining techniques. Assessing non-neoplastic pathologies in 3 and 6 months treated rats further revealed the absence of MAMAc induced cell death (Supplemental Table 3), which is typically followed by regenerative cell proliferation.…”

“…To further investigate a positive correlation between increased cell proliferation and lesion numbers, we included data sets from a previously published paralleled study, where male and female Eker rats were treated with the genotoxin aristolochic acid (AA) or the mitogenic carcinogen ochratoxin A (OTA) for 3 or 6 months (Stemmer et al 2009). Both groups shared the same controls as the MAMAc treated groups.…”

“…Continuous treatment of male and female Eker rats with 250 μg/kg BW MAMAc for up to 6 months resulted in a significant increase of pre-neoplastic and neoplastic renal lesions. However, the increase in tumor incidence after MAMAc treatment was small compared to vehicle-treated rats, especially when put in relation to the increased sensitivity of Eker rats towards other genotoxic carcinogens such as dimethylnitrosamine (Walker et al 1992) or aristolochic acid (Stemmer et al 2007; Stemmer et al 2009). Accordingly, our data suggests that only few adducts have manifested in tumor causing mutations.…”

“…Briefly, 5 days prior to sacrifice, 5 of 10 rats per group and sex were subcutaneously implanted with ALZET ® osmotic pumps (Model 2ML1, Charles River Laboratories, Germany) containing 5-bromo-2-deoxyuridine (BrdU, 20 mg/ml sterile saline, Sigma Aldrich, Germany). BrdU immunostaining was performed as described previously (Stemmer et al 2009), using a monoclonal mouse anti-BrdU primary antibody (MU247-UC, Biogenex, USA). PCNA- and BrdU-positive S-phase nuclei were quantified on randomized sections.…”

“…Eker rats carry an inherited heterozygous mutation in the tuberous sclerosis ( Tsc2 ) tumor suppressor gene and develop renal cancer at an early age (Yeung et al 1994). Numbers and incidence of cancerous lesions can be highly aggravated upon chemical renal carcinogen exposure (Walker et al 1992; Wolf et al 2000; McDorman et al 2003; Satake et al 2002; Morton et al 2002; Patel et al 2003; Stemmer et al 2007; Stemmer et al 2009). The quantitative nature of lesion formation in Eker rats therefore allows for the production of statistically powerful data after short-term exposures.…”