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Study on the Relationship between Autophagy and Anti-tumor Activitiy of Lapatinib in HER2Positive Breast Cancer Cells

Author: ZhuXingMei
Tutor: ZhangShengYong; ZhangJian
School: Fourth Military Medical University
Course: Pharmacology
Keywords: HER2positive breast cancer Lapatinib Apoptosis Autophagy mTOR AMPK 3-MA Aspirin Sodium Salicylate Lovastatin Synergisticeffect
CLC: R966
Type: PhD thesis
Year: 2013
Downloads: 38
Quote: 0
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Abstract


Breast cancer is a serious threat to the global women’s health,accounting for about a quarter of the women’s tumors. Breast cancer incidence and mortality has been significantly increasing in china recently,especially among the youth. Approximately25%of breast cancers are characterized by the amplification of HER2, whichis strongly associated with increased disease recurrence and worse prognosis. Therefore, development of molecular targeted therapeutic drugs for HER2has always been the research hotspot of this field. Trastuzumab, which is a monoclonal antibody that interferes with the HER2/neu receptor, has been widely used in the treatment of HER2positive metastatic breast cancer. However, only35%of HER2positive pa tients respond to the treatment, and resistance to the treatment develops rapidly. Therefore, it’s urgent to develop new targeted drugs against HER2.Lapatinib, a new FDA approved HER2target drug, can inhibit cancer cells proliferation by binding to the ATP-binding pocket of the EGFR/HER2protein kinase domain. Lapatinib is used in combination with capecitabine on patients who have advanced HER2-positive breast cancer. Lapatinib has given some unique advantages comparing with trastuzumab, such as oral administration, preventing both EGFR andHER2signaling pathway, penetrating the blood-brain barrier easily for treating brain metastases patients and those who has no respond to trastuzumab. However, responsiveness to the clinical treatment of lapatinib is only about24%, so whether canimprove the treatment effect through the combination with other drugs is promissing.Recent evidence has implicated that HER2-overexpressing breast cancers often showhyperactivation of the HER2/Akt/mTOR signaling pathway. Higher expression of HER2can promote over phosphorylation of PI3K, Akt, mTOR and p70SK, which facilitatestumor cells proliferation. Autophagy is a double-edged sword, which can either inhibitingor accelerating tumor cell growth. And it has been proved that cell autop hagy can beregulated through PI3K/Akt/mTOR or LKBI/AMPK/mTOR pathway. It is obscurewhethre autophagy involved in the antitumor effect of lapatinib, which is a keyproblem to be solved in our reseach.Nonsteroidal Antiinflammatory Drugs(NSAIDs) and hydroxymethylglutaryl coenzymeA reductase inhibitor (statins, HRIS) are classical drugs, who has broad spectrumantitumor action.The recent published articles on Lancet reported low doses of aspirinevery day can effectively reduced the incidence of various types of cancer, and might evenbe able to treat cancer. Lovastatin has also been shown to induce differentiation of breastcancer cell MCF-7. Both NSAIDs and HRIS have been applied in clinical practice all thetime, and the recent studies have been gradually going deep into their antitumormechanism. Aspirin has also been confirmed to inhibit colon cancer cell proliferation andmigration through inhibiting mTOR and activating AMPK pathway. So, whether NSAIDs or HRIS can show synergistic effect when combining with lapatinib treating HER2positive breast cancer will provide positive HER2breast cancer patients witheffective treatment. Therefore, we attempt to combine aspirin, sodium salicylate orlovastatin with lapatinib respectively, analyze their biological characteristics when treatingHER2positive breast cancer, and study the possible mechanism.In the first section of the dissertation, we firstly studied the antiproliferative capabilityof lapatinib on HER2positive breast cancer cells BT474and AU565by MTT, plate cloneand EDU assay. Secondly, we confirmed the apoptosis induction ability of lapatinibthrough the cell morphologic observation, flow cytometry and protein immunoblottingassay. Its effect on HER2and downstream pathways has also been examined byimmunoblotting assay. Thirdly, transmission electron microscopy, AO staining, GFP-LC3transfection and immunoblotting assay have been applied to determine the autophagyinduced by lapatinib on HER2-positive breast cancer. Then, we found that when BT474and AU565cells were pretreated by autophagy inhibitor3-MA, not only autophagyinduced by lapatinib was inhibited, but also apoptosis led by lapatinib was decreasedobviously. We also found that the cell proliferation ability enhance obviously while the Sphase arrest relief of cell cycle in condition of pretreatment with3-MA. In addition,3-MAapplication could up-regulate Caspase-3and PARP expression, compared with thetreatment with lapatinib alone. The addition of3-MA could attenuate the inhibitory role onHER2/AKT/mTOR pathway and the active role on AMPK that was raised by lapatinib.Therefore, lapatinib simultaneously induced both apoptosis and autophagy in the BT474and AU565cells, and in these settings, autophagy facilitates apoptosis. To understand thefunction of autophagy in cancer treatment has considerable merit, as well asproviding a new target for combinational therapy on HER2positive breast cancer.The second and third parts of my dissertation refer to the synergistic effect of NSAIDsor lovastatin with lapatinib combination. We try to combine aspirin, sodium salicylate orlovastatin with lapatinib respectively on treatment of HER2positive breast cancer cells.MTT assay, plate clone assay and EDU assay have confirmed that low concentration ofNSAIDs or lovastatin has synergistic anti-proliferation effect of lapatinib on BT474and AU565. When preliminary exploring combination mechanism, AO staining showed thatlow concentration of NSAIDs or lovastatin can induce autophagy on BT474and AU565cells, but when increasing concentration or action time of NSAIDs or lovastatin, the effectis given priority to killing cells rather than inducing autophagy. Therefore, autophagymay be one of the mechanisms of synergistic effect when combination of NSAIDs andlapatinib on treatment of HER2positive breast cancer cell proliferation.In summary, our study related to the mechanism of autophagy induced by lapatinib onthe treatment of HER2-positive breast cancer is still a little part of the big picture.Whenever autophagy can indeed as a novel target for HER2positive cancer treatment, thecombinational thearapy with these classical drugs such as NSAIDs or HRIS will providemore effective therapeutic regimen for HER2positive breast cancer treatment.

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