Background: Neoadjuvant chemoradiotherapy (CRT) is one of the preferred initial treatment strategies for locally advanced rectal cancer. Responses are variable, and most patients still require surgery. The aim of this study was to identify molecular mechanisms determining poor response to CRT.Methods: Global gene expression and pathway enrichment were assessed in pretreatment biopsies from patients with non-metastatic cT2-4 N0-2 rectal cancer within 7 cm of the anal verge. Downstream Akt activation was assessed in an independent set of pretreatment biopsies and in colorectal cancer cell lines using immunohistochemistry and western blot respectively. The radiosensitizing effects of the Akt inhibitor MK2206 were assessed using clonogenic assays and xenografts in immunodeficient mice.Results: A total of 350 differentially expressed genes were identified, of which 123 were upregulated and 199 downregulated in tumours from poor responders. Mitochondrial oxidative phosphorylation (P < 0⋅001) and phosphatidylinositol signalling pathways (P < 0⋅050) were identified as significantly enriched pathways among the set of differentially expressed genes. Deregulation of both pathways is known to result in Akt activation, and high immunoexpression of phosphorylated Akt S473 was observed among patients with a poor histological response (tumour regression grade 0-2) to CRT (75 per cent versus 48 per cent in those with a good or complete response; P = 0⋅016). Akt activation was also confirmed in the radioresistant cell line SW480, and a 50 per cent improvement in sensitivity to CRT was observed in vitro and in vivo when SW480 cells were exposed to the Akt inhibitor MK2206 in combination with radiation and 5-fluorouracil.

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Resistance to therapy is the major obstacle to a favorable outcome in cancer treatment. Neoadjuvant chemoradiotherapy (nCRT) can lead to complete tumor regression in a significant proportion of patients with rectal cancer (up to 40%). The possibility of avoiding radical surgery and its associated functional consequences for those who present complete response has become increasingly attractive, but approximately 60% of patients have only partial or no tumor remission after nCRT. In order to identify molecular mechanisms determining poor response to nCRT and to uncover optimized treatment strategies, we have conducted global gene expression analysis followed by pathway enrichment analysis in pretreatment biopsies from patients showing complete or poor clinical response to nCRT. We show that mitochondrial oxidative phosphorylation and phosphatidylinositol signaling pathways are consistently altered in rectal tumors comparatively. Both pathways have already been associated with resistance to radiation and chemotherapy in solid tumors and appear to converge on the activation of the Akt pathway in nonresponding tumors. To further address the role of Akt activation in response to nCRT, we evaluated by immunohistochemistry, Akt activation in an independent set of pretreatment biopsies from rectal cancer patients and observed not only high pAkt levels in those presenting poor response to nCRT but also that nuclear pAkt can also discriminate resistant tumors. We also evaluated the combination of an allosteric Akt-inhibitor MK2206 to chemoradiation in a radioresistant colorectal cancer cell line SW480 either in vitro and in vivo and observed an improvement of 50% of tumoricidal effect. Altogether, our results indicate that activation of the Akt pathway is a key event affecting response to nCRT and that combining chemoradiotherapy and Akt inhibitors such as MK2206 may significantly improve response rates to neoadjuvant therapy in rectal cancer. Citation Format: Fernanda C. Koyama, Camila M. Lopes Ramos, Jennifer M. Fernandes, Fernanda C. Ledesma, Venancio A F Alves, Fernanda C. Vailati, Angelita Habr-Gama, Rodrigo O. Perez, Anamaria A. Camargo. Akt inhibitior MK2206 combination to neoadjuvant radiotherapy: Improving the rectal cancer treatment [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A53.

Resistance to therapy is a major obstacle to a favorable outcome in cancer treatment. For rectal cancer, neoadjuvant chemoradiotherapy (nCRT) can lead to complete tumor regression in a significant proportion of patients (up to 40%) but approximately 60% of patients have only partial or no tumor remission after nCRT. A number of works searching for a differential gene expression signature has failed. As those gene signatures relies on the sample set, there is an increasing agreement that biological understanding is necessary to produce a signature in terms of pathways and biological processes. In this regard we applied pathway enrichment analysis to RNA-Seq data in order to identify biological pathways involved in tumor resistance and discriminate rectal cancer patients that is refractory to neoadjuvant treatment. Mitochondrial oxidative phosphorylation and phosphatidylinositol signaling were identified as the main pathways involved in resistance to nCRT in patients with rectal cancer. Both pathways have been reported as involved in resistance to radiotherapy and chemotherapy in solid tumors but according to the status of gene regulation observed and the literature, there is evidence that Akt pathway could be central in promoting resistance.Therefore, we evaluated the status of Akt activation by immunohistochemistry of pre-treatment biopsies of rectal tumors as well as by immunoblot of colorectal cancer cell lines utilizing antibodies against phosphorylated Akt (pS473). As a result, we observed an increased proportion of Akt phosphorylation in the resistant cell line, SW480, and in patients with TRG 0-2 in comparison to those with TRG 3-4 and in the sensitive cell line, SW48. To evaluate the implication of Akt in promoting nCRT resistance, we performed colony formation assays using Akt inhibitors as radiosensitizers. We observed an additive effect using MK2206 at sub-doses in combination to radiotherapy (2 Gy) in the resistant cell line. In fact, Akt inhibition improved in approximately 40% the tumoricidal effects of radiotherapy. Moreover, at IC50 dose this effect is even higher, reaching 60% the improvement of radiotherapy. Furthermore, the treatment of MK2206 with radiotherapy alone or in combination with 5-FU have similar effectiveness. In this setting, as Akt is involved in a range of cellular functions that promote tumor progression and metastasis, we propose that combination of Akt inhibitor and radiotherapy as neoadjuvant treatment would maximize tumor remission and improve the rectal cancer treatment. Citation Format: Fernanda C. Koyama, Camila Ramos, Angelita Habr-Gama, Venâncio Avancini Ferreira Alves, Rodrigo O. Perez, Anamaria Aranha Camargo. Implications of Akt inhibition for neoadjuvant radiotherapy: improving the rectal cancer treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 391.