In our study of 248 women with incident breast cancer from the Greater Baltimore area, increased expression of COX-2 was associated with decreased breast cancer-specific survival in patients with ER-negative and HER2-positive tumors, respectively. In patients with both ER-negative and HER2-positive tumors, increased tumor COX-2 was associated with the most inferior survival among all patient groups, as judged by the hazard ratio in the multivariable analysis. This was accompanied by increased Akt pathway activation, as judged by the phosphorylation status of Akt and two key downstream targets in the apoptosis pathway. These findings could have implications for COX-2 targeted therapy in breast cancer and suggest that patients with ER-negative and HER2-positive tumors would benefit from a COX-2 targeted therapy with the efficacy of this therapy being strongest in patients with both an ER-negative tumor status and an amplification of HER2 leading to high HER2 expression.
Two recent randomized clinical trials examined the efficacy of the COX-2 inhibitor celecoxib in combination with the aromatase inhibitor, exemestane, in postmenopausal women with hormone sensitive metastatic breast cancer [12, 18]. Both trials failed to find a significant clinical benefit with the addition of celecoxib to the exemestane regimen, despite earlier indications in small feasibility studies that there could be increased efficacy for the combination [13, 14]. Both trials were performed in predominantly hormone receptor-positive patients. HER2 status information was also available for the Falandry trial , with only 4.5% being HER2-positive. The findings in our patient population suggest that the efficacy of COX-2 inhibitors could be quite limited in ER-positive breast cancer, consistent with the trial results by Dirix et al.  and Falandry et al , suggesting that the benefit of these inhibitors as a therapeutic could be strongest in the ER-negative or HER2-positive disease. Our observation of an association between COX-2 expression with poor survival in ER-negative breast cancer is in agreement with the findings of Witton et al. , but are different to the findings of Ristimaki et al. , who found that the association of COX-2 expression with distant disease-free survival was restricted to patients with ER-positive or HER2-negative breast cancer. However, there are several differences in the evaluated patients between those in the study of Ristimaki et al and our study, which may contribute to the different findings. All breast cancer patients in our study were recruited as incident cases with surgery, and additionally our study recruited both African-American and European-American patients. The Ristimaki study (recruited 1991-1992) contained solely Finnish patients, with some of them having recurrent disease. There were also differences in receipt of therapy with 47% of patients from our study versus 61% in the Ristimaki study who received postoperative radiotherapy. Of the patients with node negative disease, 75% of our patients received either adjuvant chemotherapy or endocrine therapy, versus 9% in the Ristimaki study. There were also differences in the types of therapies received. In addition, more patients in our study had high grade disease (50%), ER-negative disease (41%) and HER2-positive disease (38%), while in the Finnish study only 30% were diagnosed with high grade breast cancer, 31% had ER-negative disease and 18% had HER2 amplification.
HER2 overexpression/amplification is an established marker of poor prognosis in both early  and late  stage breast cancer. HER2 overexpression is also associated with an increased risk of metastasis [22, 23] and a poor response to chemotherapy in the metastatic setting [24, 25]. Several therapies directed at the inhibition of HER2 are currently in use, including the recombinant humanized monoclonal antibodes against HER2, trastuzumbab (Herceptin™, Genentech), and pertuzumab (Omnitarg™, Genentech), and the small molecule tyrosine kinase inhibitor of both HER2 and EGFR Lapatinib (Tykerb™, GlaxoSmithKline) . Akt phosphorylation is associated with HER2 expression in breast cancer [27, 28], and it has been shown that HER2 transfection of MCF-7 cells leads to Akt phosphorylation mediated through the PI3K pathway . These observations are consistent with the findings in our study that Akt phosphorylation in breast tumors is significantly associated with HER2 overexpression in the tumors. Additionally Akt phosphorylation has been shown to be associated with COX-2 expression in several studies  and COX-2 specific inhibitors were found to disrupt Akt signaling in breast cancer cells . We recently demonstrated that prostaglandin E2 (PGE2), the most active pro-inflammatory metabolite of COX-2, induces phosphorylation of Akt at Ser473 and GSK-3β at Ser9, a known downstream target of pAkt . Our observation that the correlation of COX-2 with Akt pathway activation is greatest in ER-negative patients, who also have HER2 positivity, are supported by our previous finding that while PGE2 phosphorylated Akt at Ser473 and GSK-3β at Ser9 in two breast cancer cell lines, the response was considerably stronger in the ER-negative MDA-MB-231 than the ER-positive MCF-7 breast cancer cells. The result would suggest that ER-negative breast cancer cells may be more sensitive to Akt activation by PGE2 than ER-positive breast cancer cells. In summary, existing data suggest that breast cancers, which are ER-negative and overexpress both HER2 and COX-2, may have a more prominent Akt pathway activation, increased resistance to apoptosis, and a higher metastatic potential, which is all consistent with the findings in this study.
Of further interest is that HER2-positive breast tumorigenesis may be modulated in part by COX-2, and vice versa. Celecoxib was found to significantly reduce mammary tumor development in HER2/neu-induced experimental mouse models in two separate studies [31, 32]. Additionally, tumor multiplicity and size was significantly reduced in the HER2/neu transgenic mice, crossed with a COX-2-deficient background . This suggests that COX-2 significantly contributes to HER2 associated breast tumor development. Wang et al. demonstrated the ability of nuclear HER2 to transactivate COX-2 in colon cancer cells by binding to its promoter region, thus upregulating expression of COX-2 , while HER2 has also be shown to up-regulate COX-2 expression through Ras → Raf → MAPK → AP1 mechanisms in breast cancer cells . Both HER2 and COX-2 expression in breast cancer cells lead to the activation of the Akt pathway. Conversely COX-2 and its product PGE2 both lead to induction of HER2 gene and protein expression in MCF-7 breast cancer cells . These data indicate that a positive feedback loop exists between COX-2 and HER2 in breast cancer cells.
Our study has strengths and limitations. We conducted the analysis in 248 cases of incident breast cancer, recruiting both European-Americans and African-Americans allowing us to assess the impact of COX-2 on survival in both patient populations. We found that COX-2-related survival was independent of race/ethnicity, indicating that COX-2 inhibition should be equally efficacious in both patient populations. We were also able to assess the implication of the tumor p53 mutational status on the association between COX-2 and breast cancer survival and observed that COX-2-related survival was independent of the p53 mutation status. However, the existing sample size did not allow a more in-depth examination of the effect of race and p53 mutation status on survival in the context of increased COX-2. Furthermore, our immunohistochemical analysis of phosphorylated Akt cannot differentiate between the three Akt isoforms and FISH results to detect HER2 amplification were not available for this patient cohort. We also realize that some clinically important subgroups in our analysis were small, e.g. the number of patients with ER-/HER+ tumors with high COX-2 was n = 10, meaning that results for these subgroups should be interpreted with caution. Thus, additional studies are needed to further corroborate our findings.