Changes in protein expression during multistage mouse skin carcinogenesis

To directly compare the expression patterns of different proteins known to be altered during mouse skin carcinogenesis, serial sections of normal and hyperplastic skin and tumors from various stages of 7,12-dimethylbenz[a]anthracene–initiated, 12-O-tetradecanoylphorbol-13-acetate–promoted female SENCAR mice were examined by immunohistochemistry. In untreated, normal mouse skin, keratin 1 (K1) and transforming growth factor-β1 (TGFβ1) were strongly expressed in the suprabasal layers, whereas integrin α6β4 was expressed only in basal cells and only moderate staining for transforming growth factor-α (TGFα) was seen. In hyperplastic skin, TGFα expression became stronger, whereas expression of another epidermal growth factor (EGF) receptor ligand, heparin-binding EGF-like growth factor (HB-EGF), was strongly induced in all epidermal layers from no expression in normal skin. Likewise, the gap-junctional protein connexin 26 (Cx26) became highly expressed in the differentiated granular layers of hyperplastic skin relative to undetectable expression in normal skin. Expression of cyclin D1 in the proliferative cell compartment was seen in all benign and malignant tumors but not in hyperplastic skin. Beginning with very early papillomas (after 10 wk of promotion), expression of α6β4 in suprabasal cells and small, focal staining for keratin 13 (K13) were seen in some tumors. Later (after 20–30 wk), focal areas of γ-glutamyl transpeptidase (GGT) activity appeared in a few papillomas, whereas TGFβ1 expression began to decrease. Cx26 and TGFα staining became patchier in some late-stage papillomas (30–40 wk), whereas suprabasal α6β4, K13, and GGT expression progressively increased and K1 expression decreased. Finally, in squamous cell carcinomas (SCCs), there was an almost complete loss of K1 and a further decline in TGFα, HB-EGF, TGFβ1, and Cx26 expression. On the other hand, almost all SCCs showed suprabasal staining for α6β4 and widespread cyclin D1 and K13 expression, whereas only about half showed positive focal staining for GGT activity. Mol. Carcinog. 20:125–136, 1997. © 1997 Wiley-Liss, Inc.     

Smad3 regulates senescence and malignant conversion in a mouse multistage skin carcinogenesis model

Transforming growth factor beta (TGF-beta) is a growth-inhibitory cytokine for epithelial cells. In the mouse multistage skin carcinogenesis model, defects in TGF-beta 1 signaling reduce senescence in vitro and accelerate malignant progression in vivo. However, the precise postreceptor signaling pathways and specific roles played by Smad proteins in this process have not been defined. Here we show that senescence of v-ras(Ha)-transduced Smad3 null keratinocytes is delayed, whereas overexpression of Smad3, but not Smad2 or Smad4, induced senescence. The TGF-beta 1 target genes c-myc and p15(ink4b) were deregulated in the absence of Smad3. When transplanted to a graft site on nude mice, the v-ras(Ha)-transduced Smad3 null keratinocytes underwent rapid conversion from benign papilloma to malignant carcinoma, whereas wild-type keratinocytes predominantly formed papillomas. These results link Smad3-mediated regulation of growth control genes to senescence in vitro and tumor suppression in vivo.     

Alteration of Egr-1 mRNA during multistage carcinogenesis in mouse skin

Immediate early genes, including fos, jun, and early growth response-1 (Egr-1), are induced during cellular response to changes in extracellular environment. These immediate early genes are believed to mediate processes of cell growth and differentiation. In particular, Egr-1 is induced during mitogenic stimulation of a variety of cell types, including fibroblasts, B cells, and epithelial cells. In the present study, we examined Egr-1 gene expression during multistage carcinogenesis in mouse skin. After a single topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to SENCAR mouse skin, Egr-1 mRNA was induced, and maximal induction was observed at 2 h in both epidermis and dermis. Induction of Egr-1 mRNA by TPA was inhibited by fluocinolone acetonide, a potent inhibitor of tumor promotion by TPA. Egr-1 mRNA was present in primary keratinocytes derived from adult SENCAR mice. The keratinocyte cultures were maintained in low Ca(2+) medium, and Egr-1 mRNA levels became significantly elevated after the cultures were switched to high Ca(2+) medium. Additionally, a large proportion of primary papillomas and carcinomas generated from SENCAR mice by standard initiation-promotion regimens exhibited elevated Egr-1 mRNA compared with normal epidermis. Taken together, these data suggest a possible role of Egr-1 during multistage carcinogenesis in mouse skin.     

Acetic acid, a potent agent of tumor progression in the multistage mouse skin model for chemical carcinogenesis

Acetic acid, a very weak tumor promoter in the multistage mouse skin model for chemical carcinogenesis, was found to be very effective at enhancing cancer development, when applied during the progression phase of the model. Papilloma-bearing mice when repeatedly treated with acetic acid had a greater carcinoma incidence and a greater conversion of papillomas to carcinomas than vehicle treated mice. Selective cytotoxicity is discussed as a possible mechanism.     

Role of Helicobacter pylori infection in aberrant DNA methylation along multistep gastric carcinogenesis

CpG island hypermethylation is frequently found during gastric carcinogenesis. We investigated methylation profiles of p16, LOX, HAND1, THBD, p41ARC, and APC along multistep gastric carcinogenesis and determined their association with Helicobacter pylori infection. Methylation levels in these six genes were evaluated in noncancerous gastric biopsy specimens using quantitative methylation‐specific PCR in 459 patients with gastric cancer (GC), 137 with dysplasia, and 248 controls. Controls were divided into four subgroups sorted by current H. pylori infection status (active vs past or negative infection) and the presence of intestinal metaplasia (IM). In controls, active H. pylori infection significantly increased methylation levels in THBD, LOX, and HAND1 (all P < 0.001), and hypermethylation of THBD, HAND1, and APC was associated with IM. Aberrant DNA hypermethylation was correlated well with activity of H. pylori‐associated gastritis. However, methylation levels in LOX, HAND1, THBD, and p41ARC remained increased in cases with past H. pylori infection compared to those that were H. pylori negative (all P < 0.05). Hypermethylation of THBD, and possibly p16, was significantly associated with GC, regardless of the status of current H. pylori infection (all P < 0.05). These results suggest that aberrant DNA hypermethylation caused by H. pylori‐associated gastritis occurs in a gene‐specific manner along gastric carcinogenesis, which can be persistent even after the disappearance of H. pylori. Aberrant methylation of THBD might provide a link between H. pylori infection and development of GC. (Cancer Sci 2010)     

Cancer chemoprevention through interruption of multistage carcinogenesis. The lessons learnt by comparing mouse skin carcinogenesis and human large bowel cancer

Whilst in the early stages, neoplastic development is predominantly triggered by environmental genotoxic and non-genotoxic carcinogens, tumour progression becomes more and more autonomous at later stages. In this context a dysregulation of arachidonic acid metabolism seems to play a disastrous role. Conversely, non-steroidal anti-inflammatory drugs (NSAIDs) rank among the most potent and most promising agents for cancer chemoprevention probably because of their ability to inhibit prostaglandin biosynthesis, in particular, at the level of the 'pro-inflammatory' enzyme cyclooxygenase-2 (COX-2). A pathological overexpression of COX-2 resulting in excessive prostaglandin production has been found already in early stages of carcinogenesis and seems to be a consistent feature of neoplastic development in a wide variety of tissues. COX-2 overexpression is thought to occur along signalling pathways of inflammation and tissue repair which become activated in the course of tumour promotion and, due to autocrine and auto-stimulatory mechanisms, finally lead to some autonomy of tumour development (self-promotion). Prostaglandins formed along a dysregulated COX pathway have been shown to mediate tumour promotion in animal experiments and may play a role, in addition, in other processes involved in tumour growth such as angiogenesis, metastasis and immunosuppression. Moreover, genotoxic byproducts such as organic free radicals, reactive oxygen species and malondialdehyde produced in the course of prostanoid biosynthesis may contribute to genetic instability (mutator phenotype) of neoplastic cells thereby promoting malignant progression. Such mixtures of physiologically highly active mediators and genotoxic byproducts are, in addition, formed along the various lipoxygenase-catalysed pathways of arachidonic acid metabolism some of which also become dysregulated during tumour development and, therefore, provide novel targets of future chemopreventive approaches.     

DNA methylation in human lung tumors

Nucleotide profile and level of methylated cytosine (5-MC) were evaluated in DNA obtained from lung tumors in males. The level of 5-MC in DNA in malignant tissue (1.7 +/- 0.1 mol%) was half as much again that of normal lung (1.1 +/- 0.1 mol%), the difference being statistically significant.     

Effects of a biomimetic superoxide dismutase on complete and multistage carcinogenesis in mouse skin

The effects of a selective detoxifier of the proximate oxygenradical, superoxide anion, on the induction of tumors in theskin of CD-1 mice by either the initiation-promotion regimenor the complete carcinogenesis process were investigated. Theprinciple agent of interest, copper (II) (3,5-diisopropylsalicylate)₂(CuDIPS), is a low molecular weight, lipophilic copper coordinationcomplex that catalytically disproportionates superoxide anionat a rate comparable to native CuZn superoxide dismutase (SOD).The protocols used to elicit tumors were: (i) a single applicationof 0.2 µmol of 7,12-dimethylbenz[a]anthracene (DMBA) followedby twice-weekly applications of 12-O-tetradecanoylphorbol-13-acetate(TPA) in an initiation-promotion study, and (ii) either a singleapplication of 3.6 µmol DMBA followed by no further treatmentor weekly applications of 0.2 µmol DMBA in complete carcinogenesisprotocols. Application of 2 µmol CuDIPS 15 min prior tothe initiating dose of DMBA was without significant effect ontumor yield or incidence, whereas application prior to eachdose of TPA substantially reduced tumor incidence and yield.This anti-promoting property of CuDIPS can be attributed toits SOD-mimetic activity in as much as the corresponding zinccoordination complex lacking in SOD activity, zinc (II) (3,5-diisopropylsalicylate)₂,was non-inhibitory. Significant reductions in tumor yield werealso observed when CuDIPS was applied prior to DMBA in eitherof the complete carcinogenesis protocols. Additionally, covalentbinding of [³H]DMBA to epidermal DNA was markedly reduced byCuDIPS pre-treatment, suggesting that the anti-carcinogenicproperties may reflect a perturbation in superoxide anion-dependentmetabolic activation of DMBA. The induction by DMBA of ornithinedecarboxylase activity, a biochemical marker of tumor promoteractivity, was not affected by CuDIPS; however, induction ofornithine decarboxylase by TPA was potently blocked. Collectively,these effects of a biomimetic SOD further implicate reactiveoxygen species at multiple stages in chemical carcinogenesis.     

Effect of exogenous glutathione on tumor progression in the murine skin multistage carcinogenesis model

Oxidative stress has been suggested to play an integral rolein the cancer process. It may be particularly significant duringtumor progression, where there is likely to be a large amountof free radicals generated by infiltrating inflammatory cellsand dying tumor cells. In order to test this hypothesis, a varietyof free radical scavengers and antioxidants were assessed fortheir ability to inhibit tumor progression. The murine skinmultistage carcinogenesis model was used to generate papillomas,which are a population of putative precancerous lesions. Varioustest agents were applied topically to papillomas in order todetermine if they would decrease the incidence of the malignantlesion, squamous cell carcinoma. The agents tested included:reduced glutathione (GSH), butylated hydroxyanisole, vitaminE, copper(II) (3, 5-diisopropylsalicylate)₂, sodium benzoate,N-acetylcysteine and disulfiram. Under the conditions of ourexperiments, only GSH and disulfiram inhibited tumor progressionto a significant degree. Additional studies indicated that GSHprevented cancer development in a dose-dependent manner. Anotherexperiment demonstrated that when papillomas received repeatedtopical applications of diethylmaleate, a GSH-depleting agent,tumor progression was enhanced. Collectively these data suggestthat sufficient glutathione levels may be important in preventingcancer formation.     

A multihit, multistage model of chemical carcinogenesis.

Carcinogenesis involves the accumulation of genetic changes within a single cell. Tumor promotion functions in the initial clonal expansion of an initiated cell but is generally not considered to influence later stages. To investigate whether tumor promotion can influence later stages of carcinogenesis we developed a two-hit 7, 12-dimethylbenz[a]anthracene (D) protocol designed to enrich for keratinocytes that contain at least two D-induced genetic alterations. FVB/N mice were initiated with D and promoted with 12-O-tetradecanoylphorbol-13-acetate (T) or treated with acetone (A) vehicle for 6 weeks. At 7 weeks after the start of promotion, but before visible papilloma development, groups of mice were treated with a second dose of D or A and 1 week later T promotion was resumed. D/T/A/T mice developed 2.8 papillomas/mouse and D/A/D/T mice demonstrated an additive tumor response and developed 5.8 papillomas/mouse. Importantly, D/T/D/T mice developed 12.4 papillomas/mouse, thereby demonstrating a synergistic tumor response compared with D/A/D/T and D/T/A/T mice. D/T/D/T papillomas exhibited increases in suprabasal S phase cells and keratin 13 expression when compared with D/T/A/T papillomas. D/T/D/T mice developed squamous cell carcinomas (SCCs) 10 weeks earlier than D/T/A/T mice and demonstrated a 96% malignancy incidence and 1.71 SCC/mouse compared with D/T/A/T mice, which demonstrated a 28% malignancy incidence and 0.32 SCC/mouse. Greater than 90% of D/T/A/T and D/T/D/T papillomas and SCCs contained mutant Ha-ras, while a normal Ha-ras allele persisted in all cases, indicating that a gene other than the remaining normal allele of Ha-ras was a target gene for the second D hit. These data demonstrate that: (i) promotion between the first and second hits has a profound outcome on carcinogenesis, presumably by increasing the probability that a second hit will occur in a previously initiated cell; (ii) continued promotion after the second hit is required for full expression of malignancy; (iii) the classic initiation-promotion protocol can be extended to a multihit, multistage model.     

Alterations in signal transduction pathways implicated in tumour progression during multistage mouse skin carcinogenesis

The multistage mouse skin carcinogenesis model, although an artificial one, is an ideal system to study the timing of qualitative and quantitative alterations which take place during the different stages of chemical carcinogenesis, allowing analysis of the events that lead to the transition from the stage of initiation to the stage of promotion and finally to the progression of carcinogenesis. In this review we focus on the role of the H-ras gene and its target molecules during mouse skin carcinogenesis. Besides H-ras, which is a critical target of chemical carcinogens, we report alterations in oncosuppressor genes. Finally, we examine the potential suppression of metastatic dynamics of spindle cells after biological or chemical inhibition of the signalling cascades involved in mouse skin carcinogenesis.     

RUNX3 inactivation by point mutations and aberrant DNA methylation in bladder tumors

RUNX3 is inactivated at high frequency in many tumors. However, in most cases, inactivation is caused by silencing of the gene due to promoter hypermethylation. Because epigenetic silencing is known to affect many major tumor suppressor genes in cancer cells, it is not clear whether RUNX3 is primarily responsible for the induction of carcinogenesis in these cases, except for the gastric cancer cases that we reported previously. We investigated genetic and epigenetic alterations of RUNX3 in 124 bladder tumor cases and seven bladder tumor-derived cell lines. Here we show that RUNX3 is inactivated by aberrant DNA methylation in 73% (90 of 124) of primary bladder tumor specimens and 86% (six of seven) of bladder tumor cell lines. In contrast, the promoter regions of 20 normal bladder mucosae were unmethylated. Importantly, one patient bore missense mutations, each of which resulted in amino acid substitutions in the highly conserved Runt domain. The mutations abolished the DNA-binding ability of RUNX3. A second patient had a single nucleotide deletion within the Runt domain coding region that resulted in truncation of the protein. RUNX3 methylation was a significant risk factor for bladder tumor development, superficial bladder tumor recurrence, and subsequent tumor progression. These results strongly suggest that inactivation of RUNX3 may contribute to bladder tumor development and that promoter methylation and silencing of RUNX3 could be useful prognostic markers for both bladder tumor recurrence and progression.     

A mouse model of oral-esophageal carcinogenesis

Squamous cancers of the oral cavity and esophagus are common worldwide. A number of environmental factors as well as genetic alterations have been identified. However, the specific combination of genetic events and their interplay with environmental carcinogens are largely un-known. Furthermore, no good animal model existed to study the molecular changes important in the induction and progression of the disease. Here we summarize the efforts made to establish a mouse model of oral-esophageal carcinogenesis. Cyclin D1 overexpressing(L2D1+) mice were generated using an EBV promoter to specifically target the oral cavity and the esophageal squamous epithelium. Besides analyzing different environmental factors, such as nitrosamines and zinc deficiency, cyclin D1 transgenic mice were crossbred with p53-deficient mice. While L2D1+ mice exhibited a phenotype of dysplasia, different combinations of mice result-ed in invasive oral-esophageal cancer. This mouse model provides a well-defined and reproducible model of oral-esophageal cancer that should be useful for testing chemopreventive, diagnostic, and therapeutic strategies.     

Rapid induction of skin tumors in human but not mouse c-Ha-ras proto-oncogene transgenic mice by chemical carcinogenesis

The rasH2 transgenic mice carry human c-Ha-ras proto-oncogene, and are highly susceptible to chemical carcinogenesis. Previous studies showed that the mutation of c-Ha-ras induced by DMBA in the tumors of rasH2 were detected only in transgenes. To examine if the difference between the codons of the c-Ha-ras gene in human and mouse contributed to the tissue-specific sensitivity to DMBA, we generated a line of transgenic mice, mras, carrying mouse c-Ha-ras genome with its own promoter. Western blot analysis showed that the protein expression of H-RAS in the skin was increased in both rasH2 and mras compared with wild-type. Chemical skin carcinogenesis was induced by DMBA and TPA. In rasH2 mice, the latency of tumor formation was shorter than wild-type littermates. Both the number and the volume of skin tumors were increased in rasH2 than those of wild-type. However, in mras mice, enhancement of tumor formation was not observed as compared with wild-type. The mean number of tumors and the latency of tumor development was almost the same between mras and wild-type littermates. Mutational analysis showed only A to T transversion in human c-Ha-ras transgenes at codon 61 but not in murine endogenous c-Ha-ras gene in the tumors of rasH2. In the tumors of wild-type littermates and mras, A to T transversion in murine c-Ha-ras at codon 61 were detected. These results indicate that the differences in the codon of the c-Ha-ras gene between mouse and human might contribute to the tissue-specific sensitivity of DMBA.     

Expression of the TGF-beta coreceptor endoglin in epidermal keratinocytes and its dual role in multistage mouse skin carcinogenesis

Endoglin is an integral membrane glycoprotein primarily expressed in the vascular endothelium, but also found on macrophages and stromal cells. It binds several members of the transforming growth factor (TGF)-beta family of growth factors and modulates TGF-beta(1)-dependent cellular responses. However, it lacks cytoplasmic signaling motifs and is considered as an auxiliary receptor for TGF-beta. We show here that endoglin is expressed in mouse and human epidermis and in skin appendages, such as hair follicles and sweat glands, as determined by immunohistochemistry. In normal interfollicular epidermis, endoglin was restricted to basal keratinocytes and absent in differentiating cells of suprabasal layers. Follicular expression of endoglin was high in hair bulb keratinocytes, but decreased in parts distal from the bulb. To address the role of endoglin in skin carcinogenesis in vivo, Endoglin heterozygous mice were subjected to long-term chemical carcinogenesis treatment. Reduction in endoglin had a dual effect during multistage carcinogenesis, by inhibiting the early appearance of benign papillomas, but increasing malignant progression to highly undifferentiated carcinomas. Our results are strikingly similar to those previously reported for transgenic mice overexpressing TGF-beta(1) in the epidermis. These data suggest that endoglin might attenuate TGF-beta(1) signaling in normal epidermis and interfere with progression of skin carcinogenesis.     

A DNA methylation signature associated with aberrant promoter DNA hypermethylation of DNMT3B in human colorectal cancer

Altered promoter DNA methylation, one of the most important molecular alterations in cancer, is proposed to correlate with deregulation of DNA methyltransferases, although the molecular mechanisms implicated are still poorly understood. Here we show that the de novo DNA methyltransferase DNMT3B is frequently repressed in human colorectal cancer cell lines (CCL) and primary tumours by aberrant DNA hypermethylation of its distal promoter. At the epigenome level, DNMT3B promoter hypermethylation was associated with the hypomethylation of gene promoters usually hypermethylated in the healthy colon. Forced DNMT3B overexpression in cancer cells restored the methylation levels of these promoters in the healthy colon. Our results show a new molecular mechanism of aberrant DNMT3B regulation in colon cancer and suggest that its expression is associated with the methylation of constitutively hypermethylated promoters in the healthy colon.