Mechanism And Functional Research Of Autophagy Promoting Lapatinib Resistance Of HER2-Positive Breast Cancer Cells

Since late 1970 s, the global incidence of breast cancer increase a lot. The American Cancer Society released cancer statistics data on A Cancer Journal for Clinicians every year, which showing that breast cancer is still one of the most common cancers among women. Breast cancer is expected to account for about a quarter of the new estimated new cases of all women cancer types. Its mortality rate(estimated deaths) ranks the second after lung cancer.Although China is not with high incidence of breast cancer, however, in recent years the incidence rate of breast cancer increased a lot. Until 2008, the new cases of breast cancer in China accounts for 12.2% of the global, deaths accounted for 9.6%.Data released from the Chinese National Cancer Center and Bureau of Disease Prevention and Control of Ministry of Health in 2009 showed that breast cancer incidence ranked the first of female malignant tumors. Notably, however, the average diagnostic age of Chinese breast cancer patients is about 50 years old, ahead about 11 years than the average age of the western women(about 61 years old). Thus, breast cancer has become one of the most common and highest incidences of malignant tumors both in the western countries and China. Especially, the onset age of breast cancer in our country is becoming more younger, seriously affecting women’s physical and mental health.Now there are several different therapy strategies of breast cancer, including surgery, radiotherapy, chemotherapy and molecular targeting therapy etc. And the molecular targeting drugs developed most rapidly among all the fields. According to the expression levels of three different receptors of HER2(Human Epidermal growth factor Receptor-2), ER(estrogen receptor) and PR(progesterone receptor), breast cancer is classified into four different subtypes, such as Luminal A, Luminal type B, HER2 positive and triple negative.HER2 is a cell membrane receptor closely related to tumorigenesis. Its high expression will predict the worse prognosis of breast cancer patients. The statistics show that nearly a quarter of breast cancer patients are HER2 positive, and most of them with advanced malignant diagnosis. Therefore, it’s vital significance to develop the molecular drugs targeting HER2 receptor.The humanized HER2 monoclonal antibody(Trastuzumab), developed by Genentech company, can significantly extend the overall survival of HER2 positive metastatic breast cancer. Trastuzumab was approved by the United States FDA in 1998, and became the milestone of monoclonal antibody targeting drugs research. Later, FDA approved Trastuzumab as the first drug for the neoadjuvant therapy of breast cancer. However, most of HER2 positive patients acquired resistance after one year treatment of Trastuzumab, and finally caused distant metastasis.Lapatinib is a new small molecule inhibitor, which can inhibit the intracellular ATP binding sites of HER2 and EGFR, and block the downstream proliferation signal transduction. Clinical studies have confirmed that combination of Capecitabine and Lapatinib can significantly extend the time to progression(TTP) and progression-free survival(PFS). Especially, Lapatinib was still effective to breast cancer patients resistant to Trastuzumab treatment. Lapatinib targeting HER2 was approved to treat the advanced or recurrent breast cancer with combination of Capecitabine by USA FDA and China SFDA in 2007 and 2013 respectively.Studies have shown that EGF(epidermal growth factor) can activate HER2 receptor signal transduction signaling, triggering downstream signal cascade reactions. HER2 receptor activation can induce PI3 K, Akt, m TOR and p70 SK activation, and promote the proliferation of tumor cells. Lapatinib can inhibit PI3 K and Akt activation to block downstream signal transduction and the proliferation of HER2 positive breast cancer cells. However, the clinical response rate of Lapatinib is only about 24%. Primary or acquired resistance to Lapatinib severely limited its therapeutic efficacy. Therefore, the development of drug resistance is the major reason of treatment failure of Lapatinib. The resistance mechanism elucidation and effective intervention will help to further improve its clinical application.Autophagy is one of important mechanisms of cellular self-protection under extreme conditions, such as nutritional deficiencies and drug intervention etc. Autophagy can promote cell survival, but excessive autophagy induces cell apoptosis. Therefore, whether autophagy to promote or inhibit the tumor cell survival is still under controversy. Recent studies have shown that autophagy may participate in the process of tumor cells acquiring resistance. And PI3K/Akt/m TOR, LKBI/AMPK/m TOR signaling pathway might be the main upstream regulatory signaling. At the same time, the response of cellular reactive oxygen species(ROS) was also involved in the process of tumor cells to acquire resistance. To activate the cellular ROS level might be one of important mechanisms to reverse tumor drug resistance.Based on the above research background, in this subject we mainly answer the following questions:(1) Whether cellular autophagy plays important role in HER2 positive breast cancer cells acquiring resistance of Lapatinib;(2) Whether autophagy inhibition can restore Lapatinib treatment sensitivity;(3) What is the molecular mechanism of autophagy activation?(4) Compare the ROS level differences between parental and resistant cells and elucidate whether berberine can kill Lapatinib resistant cells through raising cellular ROS levels. Answering these questions will clarify the mechanism of Lapatinib resistance of HER2 positive breast cancer cells. And provide important therapeutic strategy to restore Lapatinib sensitivity.Methods and Results:1. Screening and validation of HER2 positive cell lines with Lapatinib resistance:(1) Treat HER2 positive breast cancer cell lines BT-474 and AU-565 with gradual increasing dosage of Lapatinib. And maintain for 12 months to select the stable cells with Lapatinib resistance;(2) Analyze the resistance ability of BT-474 and AU-565 cells through MTT, plate cloning and soft agar colony formation ability etc. Results: The selected resistant cells demonstrated Lapatinib resistance capacity with cell proliferation, colony formation, which is suitable for the following experiments.2. Functional evaluation of autophagy promoting HER2 positive breast cells to acquire Lapatinib resistance:(1) Whether autophagy increases in BT-474 and AU-565 resistant cells using transmission electron microscopy analysis;(2) Transfect GFP-LC3 fusion protein(autophagy biomarker) into BT-474 and AU-565 cells and then detect the autophagosome distribution using laser confocal microscope;(3) Determining the function of BT-474 and AU-565 Lapatinib-resistant cells by MTT, EDU, colony formation and flow cytometry analysis respectively;(4) Treat the Lapatinib-resistant cells with autophagy inhibitors(CQ, 3-MA and BA) and analyze the cell proliferation and apoptosis. Results: Autophagy can promote the HER2 positive cell lines acquiring Lapatinib resistance. And inhibiting autophagy may significantly enhance the sensitivity of resistant cells to Lapatinib.3. Mechanism analysis of autophagy activation in Lapatinib resistant cells:(1) Analyze the expression difference of AMPK/m TOR signaling pathway in BT-474 Par and BT-474 Lap R cells using Western blot;(2) Use AMPK phosphorylation inhibitor Compound C to block AMPK signaling pathway and analyzes its impact on drug resistant cells function. Results: The phosphorylation level of AMPK increased in BT-474 Lap R cells, suggesting AMPK activation in resistant cells, maybe the main reason inducing cell autophagy. Compound C could block the downstream signaling pathways of AMPK and the transformation from LC3-Ⅰ to LC3-Ⅱ. Thus, increase the sensitivity of Lapatinib treatment.4. Analyze reactive oxygen species status in Lapatinib resistant cells and berberine intervention research:(1) Using MTT, Ed U and flow cytometry analysis to determine whether berberine could inhibit BT-474 Lap R cells proliferation and increase apoptosis;(2) Using reactive oxygen species detection kit to analyze how berberine affecting the proliferation of BT-474 Lap R cells. Results: DCFH-DA labeling and flow cytometry analysis showed that there was less ROS in BT-474 Lap R cells. Berberine can dramatically increased the sensitivity to Lapatinib and induced resistant cell apoptosis through increasing intracellular ROS levels.Conclusion: 1. The resistant breast cancer cells of BT-474 and AU-565 had stable resistance to Lapatinib. 2. Autophagy increased in Lapatinib resistant breast cancer cells, and promoted the cells acquiring Lapatinib resistance. 3. Autophagy inhibitors could restore sensitivity of resistant cells to Lapatinib treatment. 4. Berberine can increase ROS levels of BT-474 Lap R to increase the sensitivity of Lapatinib treatment.