Preliminary Study On The Effect Of Novel Estrogen Receptor Variant ERα36 In Breast Cancer

Breast cancer is the most commonly diagnosed cancer type in women and ranks the second in female cancer deaths. American Cancer Sociaty estimates that about 194,280 new occurrences and 40,610 deaths are expected each year in the United States. The incidence rate of Chinese female breast cancer is also increasing. In certain industrialized cities, breast cancer has ranked first in female cancer incidences. Therefore, more understandings about breast cancer are needed for the improvement of early detection and comprehensive treatment.While surgery, chemotherapy, and radiotherapy have become established modalities for breast cancer treatment, endocrine therapy, such as tamoxifen and aromatase inhibitors, can provide further survival benefits with well tolerances. Among all molecular factors involved in breast cancer management, the expression status of estrogen receptor (ER) has been widely accepted as a prognostic marker and a predictor for endocrine therapy response. There are two subtypes of ER, ERa and ERβ.The 66kD ERα(here also termed ERα66), first cloned and sequenced in 1986, consists of six functional regions, from the N-terminal domain A/B to the C-terminal domain F. The N-terminal domain A/B is responsible for the ligand-independent transactivation function (AF-1). Domain C is responsible for receptor dimerization and DNA binding. Domain D is hinge region. The C-terminal domain E/F is responsible for the ligand-dependent transactivation (AF-2). Several ERβdomains show a high degree of homology to ERa, but its exact functions in breast cancer are still unclear.A single ERa66 was considered to be responsible for all ERa related estrogen biological actions in breast cancer until the discovery of its splice variants. These variants have been identified in both breast cancer cell lines. The novel 36kD variant (here termed ERa36) which was first identified and cloned in 2005 by Wang et al., lacks both transcriptional activation function domains (AF-1 and AF-2) but retains the DNA binding domain, partial dimerization, and ligand-binding domains campared to the full-length ERa66. Previous studies have confirmed ERa36 expression in breast cancer tissues and breast cancer cell lines. In human embryonic kidney cell line HEK293, it was found that ERa36 may influence ERa66 mediated transcriptional activity, but can induce the activation of the mitogenactivated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathway. Our earlier studies showed a decreased ERa36 mRNA level in colorectal cancers compared to their matched normal tissues. However, no critical study results on the effects of ERa36 in breast cancer were reported. In the present study, we compared the expression of ERa36 and ERa66 mRNA in 74 paired breast cancers and normal breast tissues, and investigated its function on cell growth and response to estrogen, tamoxifen and paclitaxel by transfecting ERa36 into ERa36-low-expression MCF-7 breast cancer cells. The details are listed below. Part One:The expression of ERa36 and ERa66 mRNA level in breast cancers and their matched normal tissues.Real-time quantitative PCR assay was applied to quantify the mRNA levels of ERa36 and ERa66 in 74 pairs of breast cancers and their matched normal tissues. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control for normalization. Besides, the findings were correlated with the clinicopathological characteristics of these breast cancer patients, including age, menopausal status, tumor size, lymph node metastasis, and tumor stage. Quantitative analysis showed that the ERa36 mRNA levels in both ERa positive and negative breast cancers were significantly lower than those in their matched normal tissues (P<0.001). The ERa36 mRNA level in breast cancers over 2 cm was significantly lower compared with cancers less than 2 cm (P=0.014). Similarly, cancers with lymph node metastasis showed significantly decreased ERa36 mRNA levels than those without lymph nodes involvement (P=0.023). The ERa36 mRNA level was also decreased in advanced diseases compared to early stage cancers (P=0.031). These results all suggest that ERa36 may associate with breast carcinogenesis and progression. The expression pattern of ERa66 in Chinese female breast cancer patients showed a close resemblance to those from African-American and Taiwanese women. Part Two:Construction and expression of ERa36 eukaryotic expression system in breast cancer cell line MCF-7 and its effects on response to estrogen, tamoxifen and paclitaxel.Our previous work had finished cloning of the full length coding region of ERa36 and had ligated it with the eukaryotic expression vector pcDNA3.1(+). However, since there is no commercial antibody against ERa36 available currently, it was not possible to confirm the successful transfection of ERa36 into breast cancer cell lines. Therefore, the coding sequence of FLAG tag was cloned and inserted into the expression vector to construct the eukaryotic expression system pcDNA3.1 (+)-FLAG-ERα36, which was verified by sequencing later. The expression vector was successfully transfected into MCF-7 breast cancer cells and the expression of ERa36 protein was confirmed by Western blot.MTT assay was performed to evaluate the influence of ERa36 on the response to estrogen, tamoxifen and paclitaxel in MCF-7 cells. At the presence of estradiol, the growth rate of ERa36 transfected MCF-7 cells was higher than those control cells which transfected with empty vectors. Suprisingly, when incubated with tamoxifen, same growth acceleration of ERa36 transfected MCF-7 cells was observed. Besides, ERa36 transfected MCF-7 cells tended to be more sensitive to paclitaxel, which showed more significant growth suppression compared to those control cells. These findings suggest that patients express relative high level of ERa36 in breast cancers are less likely to benefit from tamoxifen therapy. However, these patients are the right population for paclitaxel treatment.