Abstract
Triple-negative breast cancer (TNBC) subtype is associated with poor prognosis and a high risk of recurrence-related death in women. Despite the aggressiveness of TNBCs, targeted TNBC therapy is not yet available in the clinic. To overcome this challenge, we generated highly metastatic TNBC cells (LM) derived from metastasized lung cells via a serial spontaneous pulmonary metastasis animal model to identify targetable molecules for attenuating the progression of TNBC metastasis. Gene analysis of primary tumor (P), first-round (1LM) and second-round (2LM) metastasized lung cells revealed that mesenchymal-related genes were significantly expressed in LM cells, especially in 2LM cells. Interestingly, α9-nAChR gene expression was also dramatically induced in LM cells, confirming our previous finding that α9-nAChR plays important roles in receptor-mediated carcinogenic signals in human breast cancer development. Using α9-nAChR as a biomarker, we transfected 2LM cells with CRISPR/Cas9 lentivirus targeting the α9-nAChR genomic region (2LM-α9-nAChR-null), showing that mesenchymal markers and the migration and invasion abilities of 2LM cells were significantly attenuated in 2LM-α9-nAChR-null cells both in vitro and in vivo. In addition, the high efficiency of editing the α9-nAChR gene using a CRISPR/Cas9 lentivirus was demonstrated by gene sequencing, genomic indel frequency and protein expression analyses. Collectively, these results confirmed those of our previous study that advanced-stage breast tumors are associated with substantially higher levels of α9-nAChR gene expression, indicating that α9-nAChR expression is essential for mediating TNBC metastasis during cancer development and may potentially act as a biomarker for targeted therapy in clinical investigations.
Highlights
In 1851, Claude Bernard first discovered that nicotine directly activates muscle tissue, and nicotine was later proven to neurally stimulate common receptors through neurotransmitter release
This study suggest that α9-nicotinic acetylcholine receptors (nAChRs) expression may initiate strong signals, enhancing breast cancer metastasis during tumor development
We established a highly metastatic Triple-negative breast cancer (TNBC) cell line derived from metastasized lung cells via a serial spontaneous pulmonary metastasis animal model to evaluate significant gene alterations associated with TNBC metastasis
Summary
In 1851, Claude Bernard first discovered that nicotine directly activates muscle tissue, and nicotine was later proven to neurally stimulate common receptors through neurotransmitter release. Over the past two decades, clinical investigations have revealed that nicotinic acetylcholine receptors (nAChRs) play essential roles in mediating anti-nociception (Marubio et al, 1999), cognitive performance (Lange-Asschenfeldt et al, 2016), and oncogenic characteristics in lung, colon, and breast cancers (Dasgupta et al, 2006; Wei et al, 2009; Lee et al, 2010, 2011). In addition to a7-nAChR, the homopentameric form of α9nAChR has been reported to play important roles in promoting cancer cell proliferation (Lee et al, 2010), angiogenesis (Cooke, 2007), cancer metastasis (Wei et al, 2009), and apoptosis suppression (Paliwal et al, 2010) during carcinogenesis in response to tumor microenvironments. Some of the potential agents have even attenuated the effects of nAChR in clinical therapy (Holladay et al, 1997; Matsubayashi et al, 2004; Wang et al, 2005)
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