Estrogen receptor-positive, tamoxifen resistant breast cancer is a major obstacle for women who have undergone therapy and subsequently developed tamoxifen resistant tumors. The mechanisms by which breast cancer cells transition from tamoxifen sensitivity to resistance remain unclear. However, both preclinical and clinical studies suggest that crosstalk between the ER and growth factor and/or stress kinase signaling cascades may play a role in driving cells to tamoxifen resistance [4
]. In 2005 Guiterrez et al
. showed that in paired clinical breast cancer specimens, increased phosphorylation of p38 was positively associated with acquired tamoxifen resistance [49
]. Furthermore, Gauthier, et al
. showed that in a premalignant variant of human mammary epithelial cells, increased basal p38 activation led to increased COX-2 expression compared to control cells [62
]. COX-2 is an immediate early gene and its overexpression may be an initiating event in breast carcinogenesis [63
]. More recently, Massarweh, et al
. demonstrated in a mouse xenograft model that when ER(+) MCF-7 breast cancer cells developed a resistance to tamoxifen, the steady state levels of activated p38 were increased compared to the tamoxifen sensitive cells. Furthermore, they showed that this elevation in phosphorylated p38 could be abrogated by gefitinib, an epidermal growth factor receptor (EGFR) inhibitor [19
]. These results underscore the importance of growth factor/MAPK crosstalk with p38 in the progression of breast cancer cells to tamoxifen resistance.
In this study, we tested a novel p38 inhibitor’s ability to inhibit tamoxifen resistant breast cancer growth. Several studies have demonstrated breast cancer cells that acquire tamoxifen resistance retain some level of ER expression [66
]. To this end, we utilized the ER(+) MDA-MB-361 cell line, which is a model for acquired tamoxifen resistant and metastatic breast cancer [69
]. Using MDA-MB-361 cells, we show in vitro
that although RWJ67657 does not block phosphorylation of p38 directly, it inhibits the ability of p38 to phosphorylate and activate its downstream effector protein hsp27. RWJ67657 also exhibited potent anti-tumor properties, both in the presence and absence of estrogen, and inhibited long-term clonogenic survival. These findings demonstrate that inhibition of p38 by RWJ67657 blocks the downstream biological effects of the p38 signaling system.
Using quantitative PCR, we show that RWJ67657 inhibits the ability of p38 to stimulate transcription of Fra-1
both in vitro
and in vivo
. The fos-related antigen 1 (Fra-1) is an immediate early gene encoding a member of the AP-1 family of transcription factors and is involved in cell proliferation, differentiation, apoptosis, and other biological processes [53
]. Fra-1 transcription can be regulated by p38 [71
]. The ability of RWJ67657 to inhibit p38 and decrease Fra-1
expression is particularly intriguing because Fra-1, as an immediate early gene, seems to play a pivotal role in the process of cell transformation and carcinogenesis. Suzuki et al
showed that Fra-1 exhibited oncogenic potential in that its overexpression in rat fibroblasts stimulated anchorage-independent growth in the absence of clear morphological transformation [72
]. The inhibition of Fra-1 protein synthesis by stable transfection with a Fra-1 antisense construct significantly reduced the malignant phenotype of transformed thyroid cells [55
]. Furthermore, Fra-1 induced morphological transformation and increased in vitro
invasiveness and motility of epithelioid adenocarcinoma cells [54
]. High Fra-1 expression is also associated with a more malignant cancer cell phenotype, suggesting this gene may have a significant role in cancer progression, including that of the breast [53
]. In light of this data, a drug that can selectively knockdown expression of Fra-1 may have therapeutic promise in treating metastatic breast cancer, especially in cells that have already transitioned to tamoxifen resistance.
Given that MDA-MB-361 cells are ER(+) and p38 is known to phosphorylate and activate the ER, we tested the ability of RWJ67657 to inhibit the transcription of the progesterone receptor (PR), a known ER-mediated gene. Using a mouse xenograft model, we showed RWJ67657 was able to significantly reduce PR expression in MDA-MB-361 tumors compared to vehicle alone. We further demonstrated that RWJ67657 decreased PR expression at least in part by altering ER signaling. RWJ67657 also inhibited ER activity in an in vitro
ERE-luciferase assay. These results support the recently identified role of p38-ER crosstalk in the progression of breast carcinoma [19
The findings presented here demonstrate that p38 is crucial for breast cancer proliferation and transition from tamoxifen sensitivity to resistance. We show that the novel p38 inhibitor RWJ67657, which inhibits both p38 and ER signaling to promote its effects, shows promise as a targeted anti-cancer agent. Taken together, our results demonstrate the therapeutic potential of targeting p38 in the treatment of endocrine therapy resistant breast cancer.