The Functional Role Of The Interaction Between β2-AR And Her2 In The Malignant Progression Of Breast Cancer

The notion that psychosocial factors can affect the progression of many diseases, especially cancer, has long been suspected. Stress response can be induced by a variety of events, including trauma, family problems and bad working conditions. Stress response, which protects people from harmful stressors, is effective defensive mechanism established in evolvement. However, failure of coping with chronic stress can lead to long term and severe consequences. The data from several studies suggested that chronic stress may associate with the progression of breast cancer. For examples, the incidence of breast cancer is a three fold higher among women who are divorced or loss spouse and even nine fold higher among women experiencing extreme stress and lacking social support than normal population.Stress initiates a response of the hypothalamic-pituitary-adrenal axis (HPA), resulting in increased catecholamine level. The continuously increased catecholamine levels are observed in individuals who are under chronic stress in the recent studies. Sustained stimulation of catecholamine interferes with not only the immune system but also bio-behaviors of tumor cells. Catecholamine exerts its effects via the activation ofβ-adrenergic receptors (β-AR).β-AR plays an important role in the development and progression of tumors. However, the precise mechanism by whichβ-AR acts in tumor progression is unclear.In the first part of this study, we collected a total of 50 paraffin embedded breast cancer tissue samples and analyzed the expression of Her2 andβ2-AR by immunohistochemical staining. The results demonstrated thatβ2-AR level correlated with Her2 status in breast cancer samples. Overexpression of Her2 in breast cancer cell line MCF-7 strikingly up-regulatedβ2-AR level, impliying an intimate relationship between Her2 andβ2-AR in breast cancer cells.Her2 is a very active oncogene and overexpression of Her2 can activates multiple signaling pathways. Our data demonstrated that the constitutive phosphorylation of ERK was induced by ectopic overexpression of Her2 in MCF-7 cells and blocking of ERK signaling by PD98059, which is known to selectively block the activity of ERK, markedly inhibitedβ2-AR up-regulation induced by Her2 overexpression in breast cancer cells, implicating that ERK signaling is involved in Her2-mediatedβ2-AR expression. Further study showed that the activation of ERK up-regulated the expression of the catecholamine synthesis enzymes, and promoted the release of epinephrine from breast cancer cells. By mimicking the autocrine and paracrine of catecholamine, we confirmed that epinephrine stimulation induced the up-regulation ofβ2-AR level in breast cancer cells.Another interesting finding in this study is thatβ2-AR agonists up-regulated remarkably Her2 expression in breast cancer cells. Activation ofβ2-AR can regulate the activities of some transcriptional factors directly or indirectly. Our data showed that isoproterenol triggered the activation and nuclear translocation of STAT3 in breast cancer cell lines BT474 and MCF-7, subsequently the activated STAT3 bound to STAT3 element in the Her2 promoter and promoted Her2 expression at transcriptional and protein levels.Above data support a model whereβ2-AR and Her2 comprise a positive feedback loop in human breat cancer cells: Catecholamine stimulates the activation ofβ2-AR mediated signaling, resulting in the activation, nuclear translocation and binding to a functional STAT3 site in the Her2 promoter and thus promoting Her2 transactivaton; Her2-mediated ERK signaling induces the up-regulation ofβ2-AR expression by promoting the expression of catecholamine synthesis enzymes and autocrine of epinephrine in breast cancer cells. In this way, Her2/β2-AR feedback loop plays critical roles in the progression of breast cancer. In addition to enhancing Her2 andβ2-AR expresson, the positive feedback loop also promotes the expression of catecholamine and other cytokines involved in angiogenesis and metastasis in breast cancer cells. The comprised by Her2 andβ2-AR may play an important role in the malignant progression of breast cancer. Her2 overexpression was attributed to Her2 gene amplification. However, the recent studies revealed that Her2 gene amplification was not identified in about 80% of breast cancer patients with Her2 expression at middle level. Further investigation will reveal the novel mechanism of Her2 over-expression, and be also helpful to elucidate the mechanisms of synergism betweenβ2-AR and Her2 in the progression of breast cancer.In the second part of this study, we found that the continuous stimulation of isoproterenol could induce spontaneous lung metastasis of ovary cancer overexpressing Her2 in nude mice. By immunohistochemical analysis, we observed that the expression ofβ2-AR and Her2 frequently localized at the leading edge of the tumor. Interestingly, the nuclear localization of Her2 was found in the region whereβ2-AR was expressed highly. It strongly suggested a close relationship betweenβ2-AR over-expression and Her2 nuclear localization. Her2/β2-AR feedback loop and Her2 nuclear translocation may play an important role in the development and metastasis of breast cancer.Noticeably, isoproterenol stimulation resulted in the cleavage of Her2, producing a ~70 kDa fragment derived from Her2 C terminus, suggesting thatβ2-AR-mediated signaling was involved in the process. By analyzing the activities ofγ-secretase that cleaves the juxta-membrane region of a wide variety of transmembrane receptors, the enhancedγ-secretase activities were detected in breast and ovarian cancer cells stimulated by catecholamine. The data from bioinformatics analysis revealed that there is a potentialγ-secretase cleavage site in the juxta-membrane region of Her2 intracellula domain (ICD). Using immunofleuresent staining and confocal microscopy, we demonstrated that isoproterenol treatment induced the internalization of Her2 and nuclear translocation of Her2 ICD. Her2 in the nucleus can act as a transcriptional factor, promoting the transactivation of COX-2 gene, which was related to the lung metastasis of breast carcinoma. Our data proved that isoproterenol treatment up-regulated COX-2 expression in breast and ovarian cancer cells. Importantly, we found that chronic stimulation of isoproterenol induced spontaneous lung metastasis of Her2 over-expressing ovarian caner xenograft in nude mice. These data strongly implicated that the activation ofβ2-AR triggered the expression ofγ-secretase, by which Her2 was cleaved, and that restultant Her2 ICD translocated to the nucleus, promoting COX-2 gene expression and lung metastasis of Her2 over-expressing ovarian cancer cells in nude mice. These findings may illustrate the novel mechanisms of Her2 nuclear translocation. They may also reveal the functional roles of the trans-membrane receptors in the nucleus, cytoplasm-nucleus shortcut of signalings, and Her2 transactivation byβ2-AR in tumor progression.In conclusion, the main findings in the present study include: 1. a positive feedback loop betweenβ2-AR and Her2 in breast cancer cells; 2. Her2 cleavage and nucleus translocation of Her2 induced byβ2-AR activation; 3. the novel mechanisms of Her2 over-expression and nucleus localization; 4. the interplay ofβ2-AR/Her2 signalings in the progression of cancer.