Microenvironmental Regulation of Cancer Therapy Responses
The Axl receptor has been associated with therapy resistance and metastasis in a wide range of cancers. We have discovered that Axl-mediated drug resistance involves an autophagy-dependent cell survival mechanism in a subset of lung cancer cells, and Axl inhibition in these cells abrogates the autophagic flux and induces immunogenic cell death.Lung cancer is responsible for most cancer-related deaths worldwide. Mutations activating the epidermal growth factor receptor (EGFR) are prevalent in non-small cell lung cancer (NSCLC), and EGFR inhibitors like erlotinib are in clinical use. In spite of initial patient responses, acquired drug resistance invariably occurs, in part attributed to activation of the Axl receptor tyrosine kinase. In this project we aim to study how distinct tumor microenvironmental contexts endow cancer cells with Axl-signaling mediated phenotypic plasticity that provides resistance to anti-cancer therapeutics, and furthermore to elucidate the mechanisms by which inhibition of this signaling pathway could overcome resistance to cancer therapy. As a model of acquired drug resistance, we used the human NSCLC cell line HCC827 and erlotinib-resistant sub-clones derived from this cell line. HCC827 cells harbor an EGFR mutation (E746-A750 deletion) rendering EGFR constitutively active. The erlotinib sensitive HCC827 cells display an epithelial morphology, while the erlotinib-resistant sub-clones (HCC827 ER3 and ER10) display mesenchymal phenotypes and increased expression of Axl. Furthermore, an increase in the lipidated membrane-associated form of LC3 (LC3-II) in these cells indicates a higher autophagic flux in the erlotinib-resistant clones compared to the parental cell line. Upon Axl inhibition by the selective Axl kinase inhibitor BGB324 we observed major changes in cellular morphology and prominent cytoplasmic vacuolization. This effect was investigated by transmission electron microscopy (TEM), and in order to further examine the dynamic alterations in the autophagic degradation pathway upon Axl inhibition, we performed Western Blot and confocal imaging analysis of autophagy-related proteins. Importantly, we also observed release of damage associated molecular patterns (DAMPs), including increased extracellular calreticulin exposure, increased ATP release and increased secretion of High mobility group box 1 (HMGB1) upon Axl inhibition, which is consistent with an immunogenic cell death. These findings further support Axl as a therapeutic target in NSCLC.
Acquired drug resistance and metastasis remain the leading causes of cancer related mortalities. We have discovered that Axl-mediated drug resistance involves an autophagy-dependent cell survival mechanism in a subset of lung cancer cells, and Axl inhibition in these cells abrogates the autophagic flux and induces an immunogenic cell death.Lung cancer is responsible for most cancer related deaths worldwide, and has a poor prognosis with a 5 year survival rate of only around 15-20%. Erlotinib (Tarceva) is a drug that inhibits the EGF receptor, and is initially effective in the treatment of a subset of non-small cell lung cancer patients with activating mutations in this receptor. However, acquired drug resistance against erlotinib is a major clinical challenge, and in many cases the underlying resistance mechanism is not known. Axl is a receptor tyrosine kinase that has been associated with metastasis, drug resistance and poor clinical outcome in a wide range of malignancies, and recent results from our own laboratory and others has also linked Axl to acquired erlotinib-resistance. In this project we aim to study how distinct tumor microenvironmental contexts endow cancer cells with Axl-signaling mediated phenotypic plasticity that provides resistance to anti-cancer therapeutics, and furthermore to elucidate the mechanisms by which inhibition of this signaling pathway could overcome resistance to cancer therapy. As a model of acquired drug resistance, we have used the human non-small cell lung cancer cell line HCC827 which is sensitive to erlotinib because of an activating EGFR mutation. In addition, we have used erlotinib-resistant sub-clones of this cell line generated by two independent laboratories by in vitro exposure to erlotinib. In these resistant cells, we observed an increased Axl expression, indicating Axl as a possible driver of erlotinib-resistance. Autophagy (self-eating) is a protective mechanism where cells recycle biomolecules to conserve energy and dispose of dysfunctional organelles in order to survive and maintain tissue homeostasis. Dysregulation of autophagy is however observed in various diseases, and increased autophagy is known to promote cancer cell survival under challenging microenvironmental conditions. We observed increased expression of the autophagy marker LC3-II in erlotinib-resistant lung cancer cells, indicating a role for autophagy in drug resistance. When Axl signaling was inhibited by the selective Axl inhibitor BGB324, we observed major morphological changes, including extensive cytoplasmic vacuolization. Further investigation including Transmission Electron Microscopy, Western blot, and immunostaining supports that Axl inhibition severely abrogates the autophagic flux in the erlotinib-resistant cells. Furthermore, we evaluated the release of damage associated molecular patterns (DAMPs) upon inhibition of Axl signaling in the erlotinib-resistant cells. We observed increased ATP release, increased high mobility group box 1 secretion, and increased extracellular calreticulin exposure, which is consistent with an immunogenic form of cell death. This observation is of clinical interest because release of DAMPs and other immunostimulatory factors by tumor cells are correlated with enhanced efficacy of anti-cancer agents. Taken together, our results support further exploration of the immune-regulatory role of Axl both in tumor cells and in the tumor microenvironment, and our findings also support Axl as a valid therapeutic target in non-small cell lung cancer.
We hypothesise that distinct tumor microenvironmental contexts, comprising defined biochemical and physical parameters, endow cancer cells with plasticity traits that provide resistance to anti-cancer therapeutics. Greater insight will form the basis for new biomarkers to guide effective use of clinical targeting therapeutics to treat cancer.A confounding reality for contemporary anti-cancer treatments is the cellular heterogeneity of tumors. Tumors achieve this heritable heterogeneity through a combination of genetic and epigenetic mechanisms. As we have deepened our insight into the spectrum of genetic mutations that drive tumorigenesis and developed therapeutics against the products of specific oncogenic alleles, the epigenetic basis for treatment failures has become more apparent. Drug resistant carcinoma cells achieve phenotypic plasticity via the epithelial-to-mesenchymal transition (EMT), a normal tissue development and homeostasis epigenetic gene regulatory program. EMT is activated in response to drug treatment and immune challenge by specific microenvironmental factors. Tumor EMT is associated with chemotherapeutic resistance, immune evasion, metastasis and poor clinical outcome. The central hypothesis of this study is that distinct tumor microenvironmental contexts, comprising defined biochemical and physical parameters, endow cancer cells with phenotypic plasticity and stem cell traits that provide resistance to anti-cancer therapeutics. Lung cancer is responsible for the most cancer-related deaths worldwide. Epidermal growth factor receptor (EGFR) inhibitors such as Erlotinib (Tarceva) have been shown to be effective in non-small cell lung cancer (NSCLC) patients with distinct EGFR mutations. However, responses are rarely durable and most patients acquire resistance to EGFR targeting agents through genetic or epigenetic mechanisms. The most common resistance mechanisms are secondary mutations in the EGFR receptor, such as the T790M point mutation. However, in many cases the resistance mechanism involved is not known. Recent evidence suggests that upregulation of the Axl receptor tyrosine kinase is an important contributor to acquired resistance. We have evaluated drug responses in NSCLC cells in 2D and 3D cell culture under various microenvironmental contexts. The human NSCLC cell line HCC827 harbors a constitutively active EGFR mutation in the tyrosine kinase domain (E746-A750 deletion) that engenders sensitivity to Erlotinib treatment. HCC827 cells with acquired resistance to Erlotinib (ER3) express the Axl receptor. Drug responses to Erlotinib and the Axl inhibitor BGB324 were studied by measuring cell viability. Erlotinib-resistant cells showed an increased Axl inhibitor sensitivity when cultured at low serum and physiological oxygen levels (5 % O2, 1 % serum). When grown as 3D tumorsphere co-cultures with fibroblasts, HCC827 cells formed organized epithelial spheroids, while HCC827ER3Axl+ cells displayed mesenchymal-like invasive traits. This invasiveness was strongly inhibited by treatment with the Axl inhibitor BGB324. Our results emphasize the importance of microenvironmental influence and tumor-stroma interactions on acquired drug resistance and invasiveness in NSCLC.