釐清α9-尼古丁乙醯膽鹼受體媒介的三陰性乳腺癌進展的分子機制

Unbraid the Nicotinic Acetylcholine Receptor α9-Mediated Molecular Mechanism On the Progression of Triple Negative Breast Cancers

SONJID OCHIRBAT(SONJID OCHIRBAT)

指導教授 ： 張榮善

臺北醫學大學/醫學院/國際醫學研究碩士學位學程/碩士(2020年)

摘要

Triple negative breast cancer (TNBC) is subtype of breast cancers lacking expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptors 2 (HER2) and considered as more aggressive form with higher metastatic ability and poor prognosis. Due to the lack of surface targeted molecules, chemotherapy is the major strategy for treatments of TNBC patients, contributing to adversary side effects. Recently, several studies found that nicotinic acetylcholine receptors ɑ9 (ɑ9-nAChRs) is highly expressed on breast tumors, especially tumors derived from patients diagnosed at advanced stage. In vitro studies, it demonstrated that activation of ɑ9-nAChRs promote carcinogenic behavior including prolonging cell survival, proliferation, increased angiogenesis and metastasis. Therefore, we hypothesized that anti-ɑ9-nAChRs antibodies can be a putative therapeutic drug for TNBC treatment. In this study, we evaluated therapeutic effects of a new bispecific antibody with two antigen-binding sites against ɑ9-nAChR and methoxy-polyethylene glycol (mPEG), which provides the linkage to commercial chemotherapy drug, liposomal doxorubicin (Lipo-Dox). To characterize the therapeutic efficacy of anti a9 and anti-PEG bispecific (α-α9-αPEG) antibodies, we applied them on in vitro and in vivo models. Two animal models bearing with MDA-MB-231 human breast cancer cells were established for drug evaluation, including microtube array membrane model (MTAM) and xenograft mouse model. Comparing to the control group (buffer only) in xenograft models, tumor growth in group injected with Lipo-Dox and ɑ-ɑ9-ɑPEG conjugation drug was significantly suppressed. To assess effect of angiogenesis, ɑ-ɑ9-ɑPEG/Lipo-Dox also showed the anti-angiogenic ability by decreasing vessel density on and surrounding around MTAM membrane. Our in-vitro study results proved that bispecific antibody ɑ-ɑ9-ɑPEG significantly reduced nicotine-induced cell migration and invasion in a dose dependent manner by both transwell chamber assay and wound healing assay. Treating α9-αPEG alone inhibited nicotine-induced tube formation in human umbilical vein endothelial cells (HUVECs). The results showed that ɑ-ɑ9-ɑPEG antibody inhibits TNBC through suppressing the cancer cell proliferation, migration, invasion and angiogenesis. We suggest ɑ-ɑ9-ɑPEG bispecific antibody as a potential targeted drug in triple negative breast cancer treatment.

關鍵字

α-α9-αPEG BsAb ； MTAM ； TNBC.

並列摘要

Triple negative breast cancer (TNBC) is subtype of breast cancers lacking expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptors 2 (HER2) and considered as more aggressive form with higher metastatic ability and poor prognosis. Due to the lack of surface targeted molecules, chemotherapy is the major strategy for treatments of TNBC patients, contributing to adversary side effects. Recently, several studies found that nicotinic acetylcholine receptors ɑ9 (ɑ9-nAChRs) is highly expressed on breast tumors, especially tumors derived from patients diagnosed at advanced stage. In vitro studies, it demonstrated that activation of ɑ9-nAChRs promote carcinogenic behavior including prolonging cell survival, proliferation, increased angiogenesis and metastasis. Therefore, we hypothesized that anti-ɑ9-nAChRs antibodies can be a putative therapeutic drug for TNBC treatment. In this study, we evaluated therapeutic effects of a new bispecific antibody with two antigen-binding sites against ɑ9-nAChR and methoxy-polyethylene glycol (mPEG), which provides the linkage to commercial chemotherapy drug, liposomal doxorubicin (Lipo-Dox). To characterize the therapeutic efficacy of anti a9 and anti-PEG bispecific (α-α9-αPEG) antibodies, we applied them on in vitro and in vivo models. Two animal models bearing with MDA-MB-231 human breast cancer cells were established for drug evaluation, including microtube array membrane model (MTAM) and xenograft mouse model. Comparing to the control group (buffer only) in xenograft models, tumor growth in group injected with Lipo-Dox and ɑ-ɑ9-ɑPEG conjugation drug was significantly suppressed. To assess effect of angiogenesis, ɑ-ɑ9-ɑPEG/Lipo-Dox also showed the anti-angiogenic ability by decreasing vessel density on and surrounding around MTAM membrane. Our in-vitro study results proved that bispecific antibody ɑ-ɑ9-ɑPEG significantly reduced nicotine-induced cell migration and invasion in a dose dependent manner by both transwell chamber assay and wound healing assay. Treating α9-αPEG alone inhibited nicotine-induced tube formation in human umbilical vein endothelial cells (HUVECs). The results showed that ɑ-ɑ9-ɑPEG antibody inhibits TNBC through suppressing the cancer cell proliferation, migration, invasion and angiogenesis. We suggest ɑ-ɑ9-ɑPEG bispecific antibody as a potential targeted drug in triple negative breast cancer treatment.

並列關鍵字

α-α9-αPEG BsAb ； MTAM ； TNBC.

參考文獻

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3. Bray, F., et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2018. 68(6): p. 394-424.

4. Goldhirsch, A., et al., Strategies for subtypes--dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann Oncol, 2011. 22(8): p. 1736-47.

5. Anders, C. and L.A. Carey, Understanding and treating triple-negative breast cancer. Oncology (Williston Park), 2008. 22(11): p. 1233-9; discussion 1239-40, 1243.