The exact molecular mechanisms responsible for the onset and progression of breast cancer are still poorly understood. Unlike the majorities of solid tumors, breast cancer is usually associated with multiple small-scale genetic alterations, including minor amplifications and deletions in specific chromosomal regions. In the present work, we combined classic cytogenetic analysis (G-banding) with advanced molecular methodologies (M-CGH and BAC CGH-array analysis), as well as mathematical algorithms to find and define specific genetic changes and events leading to tumor development in DMBA-induced mammary tumors in a well-defined rat model for the disease.
G-banding analysis of 10 tumors derived from inbred SPRD-CU3 animals revealed a diploid karyotype in all. This result is in concordance with previously reported cytogenetic data for synthetic chemically (DMBA or NMU)-induced rat mammary tumors [30
], but contrasts with the reports from estradiol-induced ACI mammary tumors [32
]. M-CGH analysis of 52 tumors from three different genetic backgrounds partly confirmed this observation and showed that most of the chromosomes in the majority of tumors had profile ratios quite close to the midline, indicating a close to normal karyotype. However, recurrent segmental gains were detected in three chromosomes; RNO10, RNO12 and RNO20 (Figure ). This type of segmental gains cannot be detected by G-banding due to the limitation of this technique in detecting milder aberrations in sub group of the cells. When we studied the three tumor sets derived from the different genetic backgrounds separately (data not shown), frequency of these genetic changes appeared to vary among the tumor groups. For instance, gain of RNO12 was the most common alteration followed by RNO10 and RNO20 gains in tumor groups derived from SPRD-CU3 and the backcross animals, whereas the F1 tumors exclusively displayed gains of RNO10. The limited number of F1 tumors (six compared to 11 SPRD-Cu3 and 35 backcross tumors) might have affected this result, however, our data suggest that the nature of genetic alterations might be influenced by the genotype of the animals. In general, not many chromosome losses were detected in the tumor panel using this technique. This was most likely due to less sensitivity of M-CGH technique in detecting segmental chromosomal losses compared to chromosomal gains [34
]. Taken together, M-CGH analysis suggested a nonrandom pattern of chromosome segment gains of RNO10, RNO12 and RNO20 in the tumor material and also implied that frequency of these aberrations varied in the tumors developed in animals with different genetic backgrounds.
To obtain a higher resolution of the genetic events, we then performed BAC CGH-array analysis on a panel of 28 tumors selected based on the M-CGH data, including all 26 tumors that displayed signs of chromosomal changes and two tumors showing a normal M-CGH profile. For the purpose of this analysis, we developed an in-house BAC CGH-array platform designed to provide a high density for chromosomal regions of interest. The analysis confirmed and extended the M-CGH data and refined the affected chromosome regions to a resolution of about 150–300 Kb (i.e. the average size of the BAC clones). We subsequently derived an oncogenetic tree model based on the BAC CGH-array data, which suggested four of the observed recurrent chromosomal changes as possible initial events in mammary tumor development in this model.
The most recurrent genetic changes were as follow:
RNO5: Loss of RNO5q32 was the single most recurrent segmental alteration detected in the BAC CGH-array analysis of DMBA-induced mammary tumors (in 20 tumors, 71%). RNO5q32 loss was not detected by M-CGH due to less sensitivity of this technique in detection of segmental losses [34
]. This event was moreover placed as one of the initial events in the oncogenetic tree model, suggesting an important role for the gene(s) located at this chromosomal segment in the development of DMBA-induced mammary tumors. RNO5q32 is homologous to human chromosome 9p21 that among a number of important tumor suppressor genes, harbors the CDKN2A/2B
locus with its deletion frequently reported in different cancer types, including breast cancer [35
]. This finding was more intriguing, since M-CGH study of estradiol-induced mammary tumors in ACI rats similarly reported loss of RNO5 as the most frequently observed somatic abnormalities that usually occurred together with RNO20 losses [33
]. Our preliminary BAC CGH-array analyses of a set of ACI estradiol-induced mammary tumors confirmed and extended this finding (unpublished data). We thus conclude that deletion at this chromosome segment might represent a common signature of experimental mammary tumors (irrespective of the genetic background and the inducing agent) and thus may represent an important genetic mechanism in breast cancer development.
Remarkably, RNO5 is reported to carry mammary cancer susceptibility QTLs (Mcs5Mcstm1Emca1
, and Emca8
) in different rat mammary tumor models that control multiplicity, incidence and/or latency of tumors [7
]. Some of these QTLs are shared between strains and thus appear in different crosses, while others are specific to one cross or limited to a few crosses [7
]. In general QTLs cover large chromosome intervals and therefore identification of the gene(s) responsible for the phenotype is difficult. Recurrent RNO5q32 losses detected in this model might explain some of the reported QTLs and as the genetic changes identified in this work are at very small scale, identification of the target gene(s) might be more feasible.
RNO20: Four separate recurrent chromosomal aberrations were detected in RNO20, including three regions of recurrent gains (RNO20p11, 20p12 and 20q12) and one region of recurrent loss (RNO20q13). Together, these regions make RNO20 the most frequently affected chromosome in this investigation. In the oncogenic tree model, RNO20p11 and RNO20p12 gains were placed close to the root, but as secondary events after RNO12q11 gain and RNO5q32 loss, respectively (Figure ). The third gained chromosomal segment at cytogenetic band RNO20q12 was placed as an alternative path prior to the RNO12q11 gain and thus might represent an early and important event in the development of DMBA-induced mammary tumors. Loss of RNO20q13 was placed as one of the latest events in the RNO12q11 path and thus might be of less importance in this model. Interestingly, M-CGH analysis of ACI estradiol-induced mammary tumors [33
] showed that recurrent RNO20 losses were correlated to RNO5 deletions. In our study, we did not reach a similar conclusion; instead we found evidence for correlation of RNO20p12 gain and RNO5q32 loss in a sizable proportion of tumors. We suggest that RNO20 gains (RNO20p11, 20p12 and 20q12) might be more specific to the DMBA-induced mammary tumor model. Since these aberrations were present in a substantial proportion of experimental mammary tumors, we suggest that RNO20 gains might represent another key group of genetic changes in mammary cancer development. Gains of the homologous human chromosome regions (6p21 and 6q21) might be implicated in breast cancer development, at least in a group of patients.
RNO6q21 loss and RNO12q11 gain were the next most frequently observed chromosome aberrations in this material, each observed in 15 of the tumors (53%). In the oncogenic tree model, RNO6q21 loss was branched independently from the root, defining a subgroup of tumors independent from those with RNO5q32 deletion or RNO12q21 gain (Figure ). Our finding was more intriguing, since M-CGH analysis of ACI estradiol-induced rat mammary tumors [33
], reported similar finding of a recurrent aberration involving proximal gain and distal loss of RNO6 that never occurred in combination with RNO5 loss. Our result thus suggests that the genetic pathways (at least those involving genes located at RNO5q32 and RNO6q21) related to tumor formation in DMBA-induced rat mammary tumor are comparable to those in the ACI estradiol-induced rat model. RNO6q21 is homologous to parts of human chromosome 14q12 and 7q22. The human chromosome segment 7q22 contains an evolutionary breakpoint between the rat and human lineage. Deletion in this region is frequently reported as a primary breakpoint in cancer and has been suggested to be due to the fragility of this particular segment [39
RNO12q11 gain was found as the third initial event branching from the root in the oncogenic tree model (Figure ). This event was additionally placed as secondary event after an alternative path initiated by RNO20q12 gain in the oncogenic tree (Figure ). There were two overlapping BACs in this chromosome segment in BAC CGH-arrays, both showing gains in a substantial subset of tumors. The gene Mafk
(v-maf musculoaponeurotic fibrosarcoma oncogene family, protein K) is located in both of these clones suggesting that the Mafk
gene might be the potential target. Mafk
is known to be involved in transcription regulation and its implication has already been suggested in pancreatic cancer [40
Finally, gain of RNO10 (at 10q12 and 10q24) appeared along the pathogenic road initiated by gain of RNO12q11 in the oncogenic tree (Figure ). In all crosses, RNO10 gains were found at cytogenetic bands RNO10q12 and RNO10q24, each segment including several BACs.