Subversion of Innate Immunity Receptors Stimulation of Toll Like Receptors on Lung Carcinoma Cells Modulates Cell Survival and Response to Chemotherapy

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Lung being a site of frequent inflammation and lung cancers often developing in a context of chronic inflammation, we investigated the presence and the role of Toll Like Receptors (TLR) on lung cancer specimens from Non Small Cell Lung Cancer

(NSCLC) patients. TLR are pattern recognition receptors for pathogen-associated molecular patterns (PAMP) and endogeneous molecules released from injured and necrotic cells (DAMP) (Kumar et al. 2009). Lungs are frequently exposed to viruses such as influenza or respiratory syncitia virus, that are mainly recognized by endogenous TLR3, 7 and 8 (Kumar et al. 2009). Among the 11 different TLR described to date, we thus focused our study on TLR7 and TLR8, receptors for ssRNA (Diebold et al. 2004; Heil et al. 2004) and to a minor extent on TLR3, receptor for dsRNA (Liu et al. 2008). The stimulation of TLR7, TLR8, and TLR3 that are commonly expressed by cells of the immune system leads to the activation of NFKB and the production of proinflammatory cytokines (Napolitani et al. 2005; Hart et al. 2005; Larange et al. 2009). It induces a rapid antiviral response via the induction of type I and type II IFN which in turn enhance the adaptive immune response. Imiquimod, a TLR7 agonist is currently used topically to treat basal cell carcinoma (Tillman and Carroll 2008) or systemically in clinical trials in melanoma as immuno-stimulants and vaccine adjuvants (Dudek et al. 2007). We observed by immunohistochemistry that immune cells infiltrating NSCLC express TLR7 and TLR8 in situ, particularly within the TiBALT (Cherfils-Vicini et al. 2010).

An increasing body of evidence suggest that TLR are also expressed by nonimmune cells such as epithelial cells (Droemann et al. 2003; Tissari et al. 2005; Gribar et al. 2008) and therefore can maintain local inflammation during chronic infections. In agreement with these observations, we have detected that bronchial epithelial cells but not alveolar cells express TLR7 and TLR8 on nontumoral lung tissue sections (Cherfils-Vicini et al. 2010). Therefore, TLR7 and TLR8 may be one of the first line of defense against viruses in bronchial epithelium.

However, a close immuno-histochemical examination of tumor cells in NSCLC sections revealed that they expressed TLR7 and TLR8, at variable levels, regardless of their histological type, adenocarcinoma or squamous cell carcinoma. A first analysis of 13 tumors (8 adenocarcinoma and 5 squamous cell carcinoma) showed that all expressed TLR8 but in variable quantities; two-thirds of them were TLR7 positive, half being highly labeled. This heterogeneous expression of TLR7 and TLR8, receptors for single stranded RNA, suggested that high expressing and low expressing tumors may behave differently in the case of viral infections, or in the presence of endogenous ligands for these TLR, which could be released in the tumor microenvironment. To determine which effect could be induced by TLR7 and TLR8 triggering, we used two model cell-lines, A549 as a prototype of adenocarcinoma and SK-MES as prototypic of squamous cell carcinoma, that express TLR7 and 8. Triggering of both cell lines by Loxoribine (a TLR7 agonist), poly U (a TLR8 agonist), or gardiquimod (an agonist of both) resulted in better cell survival due to resistance to apoptosis, as assessed by a strong induction of expression of the antiapoptotic gene and protein, Bcl-2. Triggering of A549 or SK-MES by TLR7 and TLR8 agonists also induced the modulation of other genes (up regulation of CCR4 and down regulation of CD80, CD86, HLA-DR, and Fibronectin 1) which are often associated with an aggressive tumoral phenotype. The analysis of genes expressed in tumoral cells isolated from fresh tumor specimen showed that tumor cells had a transcription pattern similar to that of cell lines triggered through TLR7 and 8, suggesting that they were in an activated state in situ (Cherfils-Vicini et al. 2010).

Some patients with lung cancer are treated by neo-adjuvant polychemotherapy, consisting in platinum salts and often gemcitabine or navelbine. Both A549 and SK-MES cells stimulated by Loxoribin or Poly U were found to be resistant to chemotherapy-induced cell death. It is therefore tempting to postulate that tumoral cells which express TLR7 or TLR8 at high levels could be stimulated upon viral induced inflammation and become resistant to chemotherapy (Cherfils-Vicini et al. 2010). We are currently analyzing a cohort of lung cancer patients having received neo-adjuvant chemotherapy before surgical resection in order to assess whether high TLR7 or TLR8 expressors are less susceptible to chemotherapy than low expressors. If it were so, it would provide a novel mechanism by which tumor cells gain growth and spreading advantages, i.e., resistance to apoptosis, to chemotherapy, expression of chemokine receptors, loss of Fibronectin 1, etc. It also calls some warning on the use of TLR7 agonists as adjuvants in cancer treatment, prompting to characterize the expression of TLR7 on the tumor cells before treatment by TLR agonists. Several reports describe the expression of TLR 4 and TLR9 in lung carcinoma (Droemann et al. 2005; He et al. 2007; Ren et al. 2009). TLR expression by tumor cells of nonhematopoietic origin appears to be quite a general phenomenon as TLR2, TLR3, TLR4, TLR5, and/or TLR9 expressions have been documented in many cancer types (reviewed in Sato et al. 2009). In most cases TLR activation of cancer cells promotes survival, activates production of proinflamma-tory cytokines and chemokines, promotes angiogenesis, and therefore contributes to cancer progression. However, the response to TLR3 seems more complex and can induce opposite effects depending on the cell type. TLR3 ligation by Poly IC or Poly AU on breast cancer cells induces apoptosis in an IFNa dependent manner (Salaun et al. 2006; Andre 2005). In melanoma a proapoptotic response has been described in the presence of Poly IC as well as the induction of NFkB and production of proinflammatory cytokines (Salaun et al. 2007). We observed that the triggering of TLR3 induced apoptosis of A459 cells whereas it promoted survival of SK-MES cells. Moreover, in some cases the addition of Poly IC to chemotherapy increased sensitivity to chemotherapy-induced cell death. (Cherfils-Vicini et al. 2010).

The fact that TLR stimulation regulates cell survival and modulates their sensitivity to chemotherapy reinforces the importance of TLR expression status on tumor cells in patient's response to treatments.

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