Cells (1??104 per well) were plated in triplicate in top layers consisting of growth medium containing 0
Cells (1??104 per well) were plated in triplicate in top layers consisting of growth medium containing 0.3% agarose. managed human epidermal growth factor receptor 2 (HER2) overexpression and HER2 mediated signaling. Analysis showed that nuclear factor-kappa B (NF-B) was constitutively activated in the BT-474-R cells, a feature similar to the basal-like tumor phenotype. Pharmacologic inhibition of NF-B improved sensitivity of BT-474-R cells to trastuzumab. Interestingly, activation of HER2 impartial NF-B is not shown in luminal B breast malignancy cells. Our study suggests that by Rabbit Polyclonal to MRGX1 activating the NF-B pathway, luminal B cells may acquire a HER2+ basal-like phenotype in which NF-B is usually constitutively activated; this notion is usually consistent with the recently proposed progression through grade or development of resistance hypothesis. Furthermore, we recognized IKK-/IKK- and nuclear accumulation of RelA/p65 as the major determinants in the resistant cells. Thus our study additionally suggests that the nuclear accumulation of p65 may be a useful marker for identifying metastasis-initiating tumor cells and targeting RelA/p65 may limit metastasis of breast and other cancers associated with NF-B activation. Introduction Breast cancer is usually a heterogeneous disease consisting of unique molecular subtypes. The major subtypes are luminal A, luminal B, and triple-negative.(2) Although overall survival of breast cancer patients has improved, mortality due to metastasis still remains unchanged. The molecular mechanisms that contribute to phenotypic properties of the molecular subtypes and their relationship XMD8-92 to resistance to therapeutics remain largely unknown.(3,4) Overexpression of the human epidermal growth factor receptor 2 (HER2) occurs in approximately 20C25% of human breast cancers found on luminal A and luminal B breast cancers and is an indicator of poor prognosis for these subtypes.(5) A recombinant humanized anti-HER2 monoclonal antibody, trastuzumab (Herceptin), is approved for the treatment XMD8-92 of HER2-overexpressing breast cancer and is effective in patients with HER2+ breast cancer. However, despite the clinical benefits of these HER2-targeted therapies, almost 50% of patients with HER2+ breast cancers fail to respond to trastuzumab and the vast majority of tumors that respond to trastuzumab develop resistance within 1C2 years of treatment.(6) In some cases, the combination of trastuzumab with chemotherapy treatment improved response rates and increased overall survival rates compared to chemotherapy alone.(7C9) Unfortunately, as is the case for trastuzumab monotherapy, many patients treated with trastuzumab plus chemotherapy develop progressive disease within one year.(7C9) In particular, breast cancer patients with HER2-overexpressing, luminal B and some basal-like breast malignancy subtypes had poor prognosis post adjuvant therapy.(10) Luminal A patients display a short-term risk of relapse, but after 3 years remain stable. For luminal B patients, on the other hand, the risk of relapse occurs during the first 5 years and recurrence happens nearly 20 months post-surgery.(11) Thus, acquired resistance to trastuzumab remains an important issue in the clinical treatment of HER2+ breast malignancy. These observations suggest that there is an immediate need to address trastuzumab resistance (TZR) in patients with the relapsing luminal B breast cancer subtype and prevent metastasis-initiating tumor cells. Understanding the molecular mechanisms that contribute to the acquired resistance will ultimately allow for the identification of biomarkers that can be used to predict response to trastuzumab therapy and prevent metastasis, as well as identification of new molecular targets for the development new therapeutics. Current therapy for HER2+ breast cancer is directed at the ectodomain of the HER2 receptor.(12,13) In metastatic breast cancer, a combination treatment of trastuzumab and chemotherapeutics prolongs survival.(9) However, both and acquired resistance to trastuzumab is prevalent and overall survival gains have yet to be realized with trastuzumab monotherapy. Therefore, more effective combinations made up of trastuzumab are sought for HER2-expressing breast cancer. In addition to HER2-mediated signaling, increasing evidence suggests that other pathways, such as activation of nuclear factor-kappa B (NF-B) and STAT, are key molecular events in driving malignancies by dysregulating apoptotic, inflammatory, and immune responses.(14) Elevated levels of NF-B are frequently detected in many diseases, including breast malignancy.(15C17) The activation of NF-B in human breast cancer is confined predominantly to the XMD8-92 estrogen receptor (ER)-unfavorable subtype of cancers, particularly those that express users of the EGF family of receptors, including the EGF receptor (ErbB1) and ErbB2 (HER2/neu).(15,18) This trend was confirmed in tissue samples from patients with breast cancer.(15) Growth factor receptors are known to activate NF-B pathways following treatment of ErbB2-expressing breast cancer cells with either EGF or heregulin 1.(12,13,15,18) However, the functions of NF-B in TZR XMD8-92 breast cancers prior to exposure to cytotoxic brokers are unknown. Numerous studies suggest that several mechanisms mediate resistance to trastuzumab therapy: expression of truncated HER2 receptor (p95)(19) and epitope masking by co-receptors such as CD44, MUC4, and hylauronan.(20) However, the evolution of TZR is not fully comprehended. In an earlier study, we analyzed basal-like molecular markers in TZR patient tissues and HER2+ breast malignancy cells and found that the.