Gene expression profiling during rat mammary carcinogenesis induced by 7,12‐dimethylbenz[a]anthracene

Abstract

7,12-Dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma is a well-recognized model; however, the genetic alterations during its carcinogenesis have yet to be determined. We used laser capture microdissection to specifically isolate cells from terminal end buds (TEBs), the origin of carcinoma, at 2 weeks after sesame oil treatment (control) or DMBA treatment (DMBATEBs), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (MC). Using an oligonucleotide microarray representing 20,600 rat probe sequences, we analyzed gene expression profiles and validated mRNA and protein levels of genes of interest by real-time quantitative PCR and immunohistochemistry. The number of differentially expressed genes dramatically increased from DMBA-TEBs (63) to DCIS (798) and MC (981). Only the expression of PEP-19, an anti-apoptotic gene, showed significant increases in DMBA-TEBs (4-fold), DCIS (10-fold) and MC (16-fold). MMP-13 expression was increased markedly in DCIS (19-fold) and MC (61-fold) while OPN expression was increased 6-fold in DCIS and 8-fold in MC. MMP-7 expression was increased 4-fold in MC. Nidogen-1; a participant in the assembly of basement membranes, TSP-2; an inhibitor of angiogenesis and COUP-TFI; a transcription repressor showed significant decreases in DCIS (4-, 9-and 17-fold, respectively) and MC (10-, 37-and 100-fold). Network analyses with IPA software revealed that the most significant network included Akt groups in DCIS and ERK groups in MC. The present findings provide us with a better understanding of the molecular alteration that occur during mammary carcinogenesis and suggest the importance of PEP-19 overexpression in the very early stage of mammary carcinogenesis. ' UICCKey words: mammary carcinoma; microarray; laser microdissection; 7,12-dimethylbenz[a]anthracene; terminal end buds; ductal carcinoma in situ Increasing numbers of breast biopsies for mammographically detected microcalcifications have shifted the focus of diagnostic breast pathology from large invasive carcinomas to premalignant breast lesions. The importance of premalignant breast lesions in both risk assessment and determining patient management options makes the availability of animal models to study this aspect of disease progression highly desirable. 7,12-Dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma is a well-recognized and suitable model for studying carcinogenesis in the mammary gland. 1 Nonetheless, despite the availability of such models, relatively little is known about the molecular alterations associated with the carcinogenesis. Several studies have conducted the gene expression profiling in rat mammary glands treated with DMBA using cDNA microarrays. [2][3][4] However, all former microarray studies compared the gene expression profiles between two matched tissue such as DMBA-treated and untreated mammary glands at one time point. Furthermore, all of these data derived from bulk tissue might not adequately reflect cell-type-specific expression profiles, because bulk tissue contain...

PCP4/PEP19 promotes migration, invasion and adhesion in human breast cancer MCF-7 and T47D cells

“…Another study reported PCP4/PEP19 expression in normal and neoplastic human adrenocortical tissue where it increases aldosterone production [13, 14]. Our previous study showed that PCP4/PEP19 expression was increased in the mammary gland tissue of rats in which carcinogenesis was induced by exposure to 7, 12-dimethylbenz [a] anthracene exposure [15]. We also detected PCP4/PEP19 expression in human breast cancer and found that it exerts anti-apoptotic functions in human breast cancer cell lines via CaMKK and Akt signaling pathways [16].…”

Anti-apoptotic Effects of PCP4/PEP19 in Human Breast Cancer Cell Lines: A Novel Oncotarget

“…Recent studies have demonstrated PCP4/PEP19 expression in the normal and neoplastic human adrenocortical tissues, acting as a regulator of aldosterone production [13, 14]. However, an association between PCP4/PEP19 expression and cancer cell apoptosis has not been fully investigated yet and is largely disregarded in current discussion, even though our previous study showed that PCP4/PEP19 expression in the mammary gland tissue was significantly increased during rat carcinogenesis stimulated by 7, 12-dimethylbenz [a] anthracene (DMBA) exposure [15]. The expression of PCP4/PEP19 was significantly induced in the terminal end buds where mammary gland carcinoma develops, and the expression levels increased as cancer progressed from ductal carcinoma in situ to invasive ductal carcinoma.…”

Expression, Regulation, and Function of the Calmodulin Accessory Protein PCP4/PEP-19 in Myometrium

“…The forward and reverse primers for detection of target genes in myometrium were as follows: PCP4/PEP-19, 5 0 -GCGCGGATCCATGAGTGAGCGACAAGGTGC-3 0 and 5 0 -GGCCGTCGACCTAGGACTGACACCCAGCC-3 0 ; neuromodulin (GAP-43), 5 0 -GCAATGTTCCGTTCATCTGA-3 0 and 5 0 -CCTTAGAGCCGCAAGTTTAC-3 0 ; neurogranin (RC3), 5 0 -CACCCAAGCACACTCACTTA-3 0 and 5 0 -AACAACCC-TCTTCCCTTCATC-3 0 ; GAPDH, 5 0 -AGGGCTGCTT-TAACTCTG-3 0 and 5 0 -CTGGAAGATGGTGATGGG-3 0 . The PCR amplification was programmed as follows: denaturing at 95 C for 30 seconds, annealing at 50 C for 30 seconds (60 C for PCP4/PEP- 19), and extension at 72 C for 30 seconds. The PCR was terminated after 30 cycles.…”

“…16 PCP4/PEP-19 was also induced in models of cardiac hypertrophy 17 and altered in several cancers. [18][19][20][21][22] At the cellular level, PCP4/PEP-19 has broad effects on diverse processes, including neurite outgrowth, 23 premature neuronal differentiation, 24 long-term plasticity at neuronal synapses, 25 altered cardiac excitability and contractility, 26 and antiapoptotic activity. 27,28 PCP4/PEP-19 expression was also shown to protect against Ca 2þ overload 28 and sensitize cells to adenosine triphosphate (ATP)-dependent Ca 2þ release.…”