인간 유방암에서 HER2 유래 신호전달의 기능

Abstract

Cancer is the first major cause of death all over the world. Cancer can develop in almost any organ or tissue, such as the lung, colon, skin, bones, nerve tissue or breast. Among Korean women, the most common cancer is breast cancer. About thirty percent of breast cancers have an amplification of the human epidermal growth factor receptor 2 (HER2) gene or overexpression of its protein product. Trastuzumab (Herceptin) is an antibody that binds selectively to extracellular domain of HER2 proteins, preventing epidermal growth factor (EGF) from binding and thus it inhibits the effects of HER2 overexpression. Although Trastuzumab is currently considered one of the most effective treatments in oncology, a significant number of patients with HER2-overexpressing breast cancer do not benefit from it. Understanding the mechanisms of action and resistance to Trastuzumab is therefore crucial for the development of new therapeutic strategies. The purpose of this study was to better understand the ‘HER2-mediated intracellular signaling mechanism in human breast cancer’. Using a liquid chromatography-mass spectrometry (LC-MS/MS) proteomics approach, we found that keratin19 (KRT19) was up-regulated in HER2-overexpressing cells and tissues. KRT19 is widely used as a biomarker for the detection of disseminated tumors. KRT19 expression was induced by HER2-downstream extracellular signal-regulated kinase (ERK) at the transcriptional level. Another HER2-downstream kinase, Akt, was found to phosphorylate KRT19 on Ser35 and induce membrane translocation of KRT19 and remodeling of KRT19 from filamentous to granulous form. KRT19 was phosphorylated by Akt could bind HER2 on the plasma membrane and stabilized HER2 via inhibition of proteasome-mediated degradation of HER2. Silencing of KRT19 resulted in increased ubiquitination and destabilization of HER2. Moreover, treatment of KRT19 antibody enhanced down-regulation of HER2 and reduced cell viability. These data provide a new rationale for targeting HER2-positive breast cancers. Also, to determine the immediate role of KRT19 in breast cancer cells, we silenced KRT19 in breast cancer cells using a shRNA system. Unexpectedly, silencing of KRT19 resulted in increased cell proliferation, migration, invasion, and survival. These effects were mediated by up-regulation of Akt signaling as a result of reduced phosphatase and tensin homolog (PTEN) mRNA expression. Silencing of KRT19 decreased nuclear import of early growth response 1 (Egr1), a transcriptional factor for PTEN transcription, through reduced association between Egr1 and importin 7 (Imp7). Silencing of KRT19 increased tumor formation in a xenograft model. It was concluded that Akt activity was regulated in biphasic alteration pattern by KRT19. The cell viability was dependent on KRT19/HER2 signaling at higher KRT19 concentration and it turned to be rather dependent on KRT19/Egr1 signaling at very low/knock-out levels of KRT19. Although triple negative breast cancer [HER2, estrogen receptor (ER) and progesterone receptor (PR)-negative cancer] cells have very weak endogenous KRT19 expression, these cells also have a poor prognosis and limited therapeutic options. Therefore, it may be argued that both strong positivity of KRT19 as well as negativity of KRT19 could be used as an indicator of tumor cell survival and proliferation. Further investigation is required to determine how the two distinct mechanisms of KRT19-induced modulation of Akt activity cooperate in the heterogenous population of cells. This study also comprises one of combinatorial effects of HER2 and Notch during breast oncogenesis. Survivin down-regulates activity of caspase3/7/9 and its overexpression induces resistance to apoptosis caused by various cell death stimuli. Significantly, survivin expression is up-regulated in most human cancers, and associated with aggressive tumor and poor clinical prognosis. Notch signaling has a key role in cell-fate decision especially in cancer-initiating cells. Notch intracellular domain produced by Notch cleavage is translocated to the nucleus, and activates transcription of its target genes. In an attempt to determine the combinatory effect of HER2 and Notch signaling in breast cancer, we sought to determine the effect of HER2 on Notch-induced cellular phenomena. We first confirmed the down-regulation of Notch-dependent transcriptional activity by HER2 overexpression. Also, we confirmed that HER2/ERK signal pathway down-regulates the activity of gamma-secretase. When we examined the protein level of Notch-target genes in HER2 overexpressing cells, we observed that Notch-downstream survivin level was unexpectedly increased in HER2 cells. It was concluded that HER2 downstream signaling could enhance stability of survivin while transcription of survivin per se was decreased via Notch inhibition by HER2.