Mechanistic study of beclin1-targeting autophagy modulators to inhibit lung cancer cell proliferation

Abstract

Lung cancer is the most common cause of cancer-related death in the world. There are 2 main subtypes of lung cancer. The most common type is Non-small-cell Lung Cancer (NSCLC), which accounts for 85% of all lung cancer cases and includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. The second major subtype is Small-cell Lung Cancer (SCLC), accounting for the remaining 15%. For early-stage or localized lung cancer, curative surgery and many effective drugs are available to push the 5-year survival rate to the impressive level of 52.6%. For advanced or metastatic stage, therapeutic options are rather limited with the 5-year survival rate dropping to the dismal level of 3.5%. Novel therapeutic strategies are urgently needed. Epidermal Growth Factor Receptor (EGFR) is a major drug target for non-small-cell lung carcinoma (NSCLC). Tyrosine Kinase Inhibitors (TKIs) like erlotinib are potent inhibitors of EGFR and have achieved impressive clinical success against NSCLC. However, NSCLC cells readily develop resistance to TKIs by acquiring mutations in EGFR or other oncogenes. Novel strategies to inhibit EGFR are needed to overcome this urgent problem of TKI resistance. SCLC is characterized by highly proliferative and metastatic lung cancer, making it ineligible for surgery in most cases. Current outcome of clinical treatment for SCLC is poor since lack of known predictive or diagnostic biomarkers and poor response to standard therapies. SCLC is usually sensitive to initial chemotherapy but prone to recurrence and acquired drug resistance. Upon receiving treatment, the median survival time (MST) of SCLC patients is only 8-20 months(Puglisi, Dolly et al. 2010). Therefore, it is challenging but necessary to develop effective therapeutics for SCLC. Autophagy is an evolutionarily conserved catabolic process for recycling or degrading organelles and cellular macromolecules and plays important roles in regulating cell survival and death. Autophagy acts like a double-edged sword in cancer and exerts both positive and negative effects in a context-dependent manner. It was reported that inhibiting autophagy could suppress lung cancer progression, especially at the early stage, making autophagy is an interesting and potential target for cancer therapy. Our lab designed a series of all-hydrocarbon stapled peptides (Tat-SPs) targeting at interfere with Beclin 1 self-association to promote the interaction between Beclin 1 and Atg14L/UVRAG. Here I present data to show that one lead peptide Tat-SP4 upregulated autophagy activity, and promotes two overexpressed membrane proteins, EGFR and c-Met, endolysosomal trafficking as well as inhibits the downstream signaling pathway. Tat-SP4 shows potent anti-proliferative efficacy in different lung cancer cell lines and exhibit synergistic effect with EGFR-TKIs in NSCLC cell lines. The cell death induced by Tat-SP4 could be rescued by digoxin, a cardioglycoside that blocks the Na+/K+ ATPase and is accompanied by the morphological features similar to that of autosis. We also found that Tat-SP4 partially depolarized mitochondria membrane potential, increased intracellular reactive oxygen species, inhibited the mitochondrial OXPHOS activity and induced mitochondrial permeability transition pore (mPTP) opening. Meanwhile, intracellular calcium homeostasis was disrupted when lung cancer cells were challenged by Tat-SP4, which is characterized by a release of Ca2+ from ER followed by increase in Ca2+ level in mitochondria and cytoplasm. In SCLC cell-based xenograft model, Tat-SP4 significantly inhibited tumor growth without obvious toxicity. In conclusion, our novel designed Beclin-1 targeting stapled peptides have shown potent anti-proliferative efficacy in lung cancer model both in vitro and in vivo, suggesting that our study provides a new strategy to target autophagy for lung cancer treatment. Our stapled peptides may offer an approach orthogonal to the existing regimen of tyrosine kinase inhibitors (TKIs) that directly inhibit the kinase activity of EGFR. Thus, our stapled peptides may potentially serve as the prototype for a new class of therapeutic agents that can be used in conjunction with existing therapies toward better treatment for lung cancer.