Abstract LB-430: Hedgehog signaling is required for the survival and tumorigenicity of cancer cells.
Authors: Singh Samer , Wang Zhiqiang , Giambelli Camilla , Long Jun , Briegel Karoline J. , Bejarano Pablo A. , Capobianco Anthony J. , Robbins David J. 
Published: July 2012

Background: Using small molecule smoothened (SMO) antagonists, Hedgehog (HH) signaling had been implicated to play an important role in a variety of cancers (e.g. Brain, Lung, Pancreas, digestive tract etc.). Recently, based on studies in a selected set of pancreatic and colorectal cancer cells it has been suggested: a) earlier observation of HH signaling dependence of various cancers by others and us is due to non-specific effect of SMO antagonists; b) HH producing cancer cells do not have a functional HH pathway; c) HH signaling is not required for the growth or survival of cancer cells rather it affects the growth of tumor by activating HH pathway in stroma cells. Given the reported prevalence of HH signaling in cancers and the suggested implication of this latter work on devising a successful cancer therapy, we decided to investigate the role of HH signaling in cancer in a manner independent of SMO antagonist. Lung cancer, being one of the deadliest cancers with the reported higher incidence of HH signaling, was used as a test system to evaluate the basic assumptions of this recently proposed model of HH role in cancer.

Objective: Define the role of HH signaling in cancer cells.

Methodology: The human non-small cell lung carcinoma (NSCLC) cell lines (HOP62, A549, U1752, H23, H157, H522) were used as a model system to study the relevance of HH signaling in cancer cells. The HH signaling was modulated by shRNA mediated knockdown of various HH signaling components (e.g. SMO, GLI1, SHH). The lentiviruses expressing shRNA were made and used to deliver the shRNA in NSCLC cells. HH responsiveness (quantitative real time RT-PCR and western blotting), cell proliferation (ATP quantitation), survival (Annexin-V labeling), anchorage independent growth (soft agar assay) and tumorigenesis (xenografts in Nu/Nu nude mice) were evaluated following shRNA mediated attenuation of HH signaling. Currently, HH pathway activation in cancer and stroma cells of primary lung cancer samples is being evaluated by immunohistochemistry (IHC) and RNA in situ hybridization (ISH).

Preliminary Results and Conclusions: Exogenous expression of SHH in HOP62 and A549 induced the HH target gene GLI1 and PTCH1 expression while shRNA mediated knockdown of HH pathway components (e.g. SMO, SHH) inhibited the HH target gene expression. Further more, knockdown of HH pathway components, attenuated the proliferation, increased the apoptosis while decreased the anchorage independent growth of tested lung cancer cell lines. GLI1 shRNA mediated attenuation of HH signaling in A549 cells, greatly reduced their tumorigenicity in nude mice. We anticipate seeing activated HH signaling in cancer and stroma cells of primary lung tumor samples by IHC and ISH. Together, these results would indicate that cancer cells do elaborate a functional HH signaling pathway and require HH signaling for growth, survival and tumorigenicity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-430. doi:10.1158/1538-7445.AM2011-LB-430