ABCG2, or breast cancer resistance protein (BCRP), is the second member of the G subfamily of the ATP-binding cassette (ABC) efflux transporter superfamily that has been the subject of intense study since its discovery two decades ago. In 1998, Doyle et al. cloned the gene responsible for the novel resistance phenotype from the MCF-7 Adr/Vp subline in the absence of P-gp or MRP1. They named the gene BCRP for breast cancer resistance protein since it was cloned from a breast cancer subline. Soon after, two nearly identical transporters, termed ABCP for the ABC transporter highly expressed in placenta  and MXR for the mitoxantrone resistance gene, were found in different labs. When the sequences for the genes BCRP/ABCP/MXR became available, they proved to be nearly identical. The Human Genome Nomenclature Committee assigned the gene the name ABCG2, making it the second gene in the G subfamily of ABC transporters that is made up of only half-transporters.
The human ABCG2 gene is localized on chromosome 4, band 4q21-4q22 in normal cells . It extends over 66 kb containing 16 exons and 15 introns, and the resulting protein is 655 amino acids long . The development of antibodies to ABCG2 enabled the detection of ABCG2 in formalin-fixed, paraffin-embedded tissues. Maliepaard et al. examined ABCG2 expression in normal tissues using the BXP-21 and BXP-34 monoclonal antibodies. ABCG2 expression was found in placenta, particularly in the synctiotrophoblastic cells, as well as in colon, small intestine, biliary canaliculi, breast tissue, venous endothelium, and in capillaries .
In the present study, we studied ABCG2 expression in paraffin-embedded tumor samples using the IHC method with the BXP-21 antibody, and the positive stain for ABCG2 is located in the cytoplasm and cytomembrane of cells as the product leaflet of the antibody indicates. The expression of ABCG2 in the normal mammary glandular epithelia and in the stromal cells is consistent with previous literature.
In solid tumors, Diestra et al. studied ABCG2 expression in paraffin-embedded tumor samples with the BXP-21 antibody and reported frequent expression in tumors of the digestive tract, endometrium, lung and melanoma . Breast cancer has been most extensively studied, with most reports concluding that ABCG2 expression was relatively low and did not appear to correlate with clinical outcome in the studies of Kanzak et al.  or Faneyte et al. .
Inconsistent with the above reports, in our study, the expression of ABCG2 in most tumor cells (86.7%) of the breast invasive ductal carcinoma is positive, in which about 40% cases present relatively high ABCG2 expression in the breast cancer cells. Furthermore, the expression of ABCG2 is correlated with the expression of HER2 significantly (p = 0.001) by means of statistic analysis of 196 breast cancer cases detected by IHC. Those patients with high expression of ABCG2 were demonstrated to be more frequently showing high immunoreactions with HER2, which suggests that ABCG2 may have some correlation with worse biological behavior and clinical aggressiveness. Besides, the expression of ABCG2 is correlated with lymph node metastasis (p = 0.049) and clinical stage (p = 0.015), which suggests that the patients with high expression of ABCG2 may have a worse prognosis than those with low expression of ABCG2.
In the previous reports, much fewer cases (43 cases and 52 cases, respectively) were examined than that in the present study (196 cases). They mainly detected the levels of BCRP mRNA in those cases, and only 27 cases of breast cancer underwent IHC testing to examine the expression of BCRP protein [7, 8]. These may explain the differences between their studies and the present one. To the best of our knowledge, this is the first report that the expression of ABCG2 is correlated with some clinicopathological parameters and biological markers in the cohort of breast invasive ductual carcinoma. We think it is of great importance for further research.
As we all know, HER2 is an oncogene that has been studied extensively. Human epidermal growth factor receptor 2 (ERBB2, formerly HER2/neu, c-erbB2), 1 of a family of 4 membrane tyrosine kinases (TKs), was found to be amplified in a human breast cancer cell line 26 years ago , and this amplification was shown to be important in the pathogenesis and progression of human breast cancer . Since that time, HER2 amplification and resultant HER2 protein overexpression have been linked to important tumor cell proliferation and survival pathways, several drugs have been developed to target the pathway, and the detection of HER2 has become a routine prognostic and predictive factor in breast cancer.
Normal tissues have a low complement of HER2 membrane protein. Overexpression of HER2 is seen in 20% of breast cancers, and it confers worse biological behavior and clinical aggressiveness in breast cancer [22, 24]. The differential in HER2 expression between normal tissues and tumors helps to define HER2 as an ideal treatment target. Trastuzumab, the first treatment targeting HER2, is well tolerated in patients and has little toxicity because its effects are relatively specific for cancer cells over expressing HER2.
Thus, the significant correlation (p = 0.001) between the expression of ABCG2 and of HER2 may suggest that ABCG2 is not only an ABC transporter which plays a role in drug-resistance of breast cancer chemotherapy but also a novel potential bio-marker which can predict biological behavior, clinical progression, prognosis and chemotherapy effectiveness. And it indicates that when the tumors are treated with chemotherapy and targeting medicines, the antagonists of ABCG2 are supposed to be applied together.
Because the activated AKT mediates the metastasis of tumor cells by the HER2/PI3K/PTEN/AKT pathway , HER2 has direct correlation with lymph node metastasis of breast cancer. Due to the strong correlation between the expression of ABCG2 and of HER2, it is easy to understand the correlation between ABCG2 and lymph node metastasis status (p = 0.049) found in this study. Clinical stage is dependent on the lymph node metastasis in large extent, and the correlation between them is obvious. So the significant correlation (p = 0.015) between the expression of ABCG2 and clinical stage found in the present study is pleasant to be accepted by us. Because there was no case in clinical IV stage in the 196 patients which were used in the study, it is not possible to evaluate the correlation between ABCG2 expression and cases with distant metastasis.
Two functional elements in the ABCG2 promoter, the estrogen  and hypoxia  response elements, and a peroxisome proliferator-activated receptor g (PPARg) response element upstream of the ABCG2 gene  have been shown to control ABCG2 expression. So it seems that the expression of ER should be correlated with ABCG2, but the result of our study failed to show this. It is not disappointing, because previous studies have showed that the effects of E2 on ABCG2 expression in breast cancer cells were inconsistent and tissue-specific as a result of the differences between ERαand ERβ. In several human breast cancer cell lines, E2 exposure decreases BCRP protein expression and function, but it does this by acting through ERαand not ERβ. However, E2 has also been reported to increase BCRP protein expression in a human breast cancer cell line by signaling through ERα. In a human placenta cell line, E2 signaled through ERβ to up-regulate BCRP ; and Anika M S Hartz et al. found that E2 signals through ERβ, PTEN/PI3K/Akt/GSK3 to down-regulate the expression of BCRP . Thus, both ERαand ERβ can be involved in E2 regulation of BCRP, but the signals involved and the effect on BCRP (up- or down-regulation) seem to be inconsistent and tissue-specific. These may be the reason why the correlation between ABCG2 and ER is not significant (p = 0.136).
As to the possible regulation signaling pathway between HER2 and ABCG2, we speculate some possibilities below. A recent study by Bleau et al. demonstrated that PTEN/PI3K/Akt signaling regulates ABCG2 activity in mouse and human gliomas , and PTEN/PI3K/AKT pathway has already been confirmed be one of the signal-transmitting pathways in the HER signaling network. HER2 partners with HER3 to form heterodimerization, and HER3 has multiple docking sites for PI3K. So, HER2 partnering with HER3 is the most potent stimulator of the PI3K/AKT pathway .
The HER2/PTEN/PI3K/AKT/ABCG2 pathway may be the signaling regulation mechanism between HER2 and ABCG2. Of course, all the above speculated regulation pathways need to be certificated by further study.