Cyclin-dependent kinase 1 targeting improves sensitivity to radiation in BRAF V600E colorectal carcinoma cells.

Preoperative chemoradiation is currently the standard of care in locally advanced rectal carcinoma, even though a subset of rectal tumors does not achieve major clinically meaningful responses upon neoadjuvant chemoradiation. At present, no molecular biomarkers are available to predict response to neoadjuvant chemoradiation and select resistant tumors willing more intense therapeutic strategies. Thus, BRAF mutational status was investigated for its role in favoring resistance to radiation in colorectal carcinoma cell lines and cyclin-dependent kinase 1 as a target to improve radiosensitivity in BRAF V600E colorectal tumor cells.Colony-forming assay and apoptotic rates were evaluated to compare the sensitivity of different colon carcinoma cell lines to ionizing radiation and their radiosensitivity upon exposure to BRAF and/or cyclin-dependent kinase 1 inhibitory/silencing strategies. Cyclin-dependent kinase 1 expression/subcellular distribution was studied by immunoblot analysis.Colon carcinoma BRAF V600E HT29 cells exhibited poor response to radiation compared to BRAF wild-type COLO320 and HCT116 cells. Interestingly, neither radiosensitizing doses of 5-fluoruracil nor BRAF inhibition/silencing significantly improved radiosensitivity in HT29 cells. Of note, poor response to radiation correlated with upregulation/relocation of cyclin-dependent kinase 1 in mitochondria. Consistently, cyclin-dependent kinase 1 inhibition/silencing as well as its targeting, through inhibition of HSP90 quality control pathway, significantly inhibited the clonogenic ability and increased apoptotic rates in HT29 cells upon exposure to radiation.These data suggest that BRAF V600E colorectal carcinoma cells are poorly responsive to radiation, and cyclin-dependent kinase 1 represents a target to improve radiosensitivity in BRAF V600E colorectal tumor cells

Molecular mechanisms responsible for radiation resistance in colonrectal tumors

Abstract Introduction Radiotherapy is a well-established therapeutic modality for cancer. It is considered a crucial treatment for most common types of cancer and is usually used in conjunction with chemotherapy, hormone therapy or surgery. However, the presence of radioresistant cells is one of the major obstacles to successful treatment with radiotherapy. Ionizing radiation exerts its cytotoxic effect by the induction of double strand breaks (DSBs) and non-DSB highly clustered DNA lesions consisting in a combination of single strand breaks (SSBs), abasic sites and oxidized bases within 5–10 base pairs. Radiation is known to activate multiple signaling pathways, causing cancer cells to become inactivated and resulting in diverse types of stress responses, including apoptosis, cell cycle arrest, senescence and gene induction. However, a large number of tumours fail to respond to radiotherapy as they are less sensitive or more resistant to radiation. Various studies on the molecular mechanisms of resistance to radiotherapy have been carried out. However, obstacles related to overcoming this resistance remain to be solved. Therefore, identification of the radiation-responsive genes may aid to better understand the molecular mechanisms involved in the response of tumours to radiation and, ultimately, improve radiotherapy. We focused our attention on colorectal cancer (CRC), which is one of the three leading causes of cancer deaths worldwide. Most colorectal cancers are sporadic, with dietary risk factors implicated in their development. Despite curative surgery, patients still have a significant probability of disease relapse and poor survival. Much interest has been generated in the last years in neoadjuvant treatment that would improve operability and prevent recurrent diseases. Neoadjuvant chemoradiotherapy is an area of active research in rectal cancer. Indeed, outcomes of patients with rectal cancer have improved over the last decade, but this benefit has not extended to all subtypes of this disease. Many trials have been conducted to improve the outcome and decrease recurrence possibility. It is without doubt that tracing the underlying molecular mechanisms within the adopted strategy for cell death is a cornerstone for the success of such trials. The purpose of our study was to evaluate if inhibition of determinate key points could enhance radiosensitivity in colorectal cancer cell lines. Materials and Methods Patients (pts) and neoadjuvant therapy. Between October 2006 and December 2013, 116 pts with locally advanced or distal T2 rectal tumours were treated by chemoradiation followed by surgery. Median follow-up was 66 months (IQR 56–73) for all pts. Cell lines. We used cells of human colorectal adenocarcinoma: COLO320 (RAS and BRAF wild-type), HCT116 (RAS-mutated), HT29 (V600E BRAF mutated). Cell lines were supplied by the American Type Culture Collection (ATCC). Chemotherapeutic. 5-fluorouracil (5-FU), commonly used to treat colorectal malignancies in association with radiotherapy. 5-FU was used at concentration of 5-500 nM. Inibitors. PLX4720, a potent and selective inhibitor of the V600E mutant form of the B-Raf protein, and HSP990, a dual HSP90/TRAP1 inhibitor. Gene silencing by siRNA. Cells were transfected with BRAF-targeting siRNA, TRAP-targeting siRNA or nontargeting siRNA. Cell culture. Cells were seeded in a 6-well plate at a density of 300 and grown in a medium containing 10% heat-inactivated fetal bovine serum. The cells were maintained in a humidified atmosphere containing 5% CO2 at 37˚C. Irradiation. A preliminary dosimetric study was necessary. Plates with cell lines were CT scanned. The CT data were imported into a treatment planning system and elaborated allowing isodose coverage of 95–107%: 1 cm plexiglas layer was used for radiation dose build-up, due to its tissue equivalent characteristics. Radiation was carried out at room temperature and delivered with 1.8-24 Gy, using a therapeutic linear accelerator (Elekta Sinergy) and 6-MV X-rays. Cytofluorimetric Assay. After irradiation, cell lines were labeled using the Annexin V-FITC (fluorescein isothiocyanate) / 7-AAD (7-amino-actinomycin D) kit. Apoptosis was assessed by flow cytometric analysis of Annexin V and 7-AAD positive cells. Clonogenic Survival Assay. Cell were grown in standard medium for about 2 weeks after irradiation to assess the capacity to form colonies. Cells were fixed with cold methanol for 25 minutes and stained with 1% crystal violet. The number of colonies containing at least 50 cells was determined. Surviving fractions for each treatment were determined by normalizing the average plating efficiency for each dose to the plating efficiency at 0 Gy. Western Blot. Cell lysates were prepared using buffer containing phosphatase and protease inhibitors and protein concentration was determined. Equal amounts of protein were subjected to electrophoresis performed under reducing conditions and gels were blotted to nitrocellulose membranes. All western blotting experiments were conducted in duplicate. Statistical analyses. Comparison of the relative sensitivity of the cell lines was conducted using Analysis of Variance for three or more groups. Unpaired t-tests were used for comparisons of cytotoxicity between two conditions or cell lines. Results and Discussion Although current total mesorectal excision is curative for small tumours, the risk of locoregional recurrence, distant metastasis and death increases with tumours extending through the muscularis propria (T3 or T4) or nodal involvement (N1 or N2; stage II and III tumors). One strategy to minimize recurrence in patients with rectal cancer is neoadjuvant chemoradiotherapy. Preoperative ionizing radiation downstage tumours and is well tolerated. Ionizing radiation, however, results in a wide spectrum of clinical response and the magnitude of benefit is heterogeneous. We analyzed our cohort of pts with locally advanced or distal T2 rectal tumours and we observed 21% pathological complete responses, 39% partial responses and 40% stable diseases. The estimated 2-year disease-free survival was 91.0% (95% CI 84.8-97.6). The estimated 2-year overall survival 96.2% (95% CI: 92.0-100). One of the hallmarks of cancer cells is the up-regulation of cellular pathways that provide survival advantages by promoting proliferation and/or decreasing cell death. We evaluated the hypothesis that the RAS/RAF mutational status may influence cell response/resistance to radiation/chemoradiation. Upon analysis of clonogenic ability and apoptotic response to radiation in HCT116, COLO320 and HT29 cell lines, COLO320 cells showed sensitivity to radiation, while HT29 cells (BRAF-mutated) revealed to be the more radioresistant and HCT116 cells (KRAS-mutated) an intermediate phenotype. Our data suggest that resistance to ionizing radiation is mediated by activation of the Ras/MAPK pathway. Such pathway is the Ras/Raf/mitogen activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathway, which is involved in cell proliferation, differentiation, apoptosis, and survival. CRCs frequently exhibit activation of the Ras/MAPK pathway via activating mutations in Ras and/or Raf. The presence of either a Ras or Raf mutation is associated with an inferior prognosis compared to non-mutated tumours. Less than 10% of patients with metastatic CRCs have tumours with a point mutation in BRAF, a component of the RAF/MEK/ERK signaling pathway. Similar to other cancers, more than 95% of the BRAF mutations in CRC affect the V600 position of the protein, resulting in constitutive RAF/MEK/ERK pathway activation. Inhibition of the Ras/MAPK pathway has also been exploited as a means to sensitize tumours cells to cytotoxic chemotherapy. As some studies hypothesized, in our experience inhibition of signaling via the Ras/MAPK pathway enhances sensitivity also to radiation. To assess if specific inhibitors could enhance the radiation sensitization observed with 5-FU, we performed clonogenic survival assays with three tumour cells lines. Doses of X-rays and inhibitors and timing of 5-FU were chosen based on published data and preliminary work performed in our laboratory confirming radiation sensitization. In all cell lines the combination of 5-FU and radiation showed to arrest cell growth to a greater extent compared to radiotherapeutic treatment alone. However, HT29 cells were confirmed to be more resistant than COLO320 and HCT116 cells also to the combination of 5-FU and radiation. Inhibition of RAS/RAF/ERK pathway was evaluated as a strategy to sensitize rectal cancer cell lines to radiation. This was achieved by specific BRAF inhibitors, BRAF silencing and HSP990, a dual inhibitor of HSP90 and TRAP1. The inhibition of B-Raf protein with PLX4720 showed a moderate but not significant sensitizing effect, according to literature results. Conversely, BRAF siRNA silenced cells resulted significantly more sensitive than control and negative transfected cells. HSP990 significantly increases the radiosensitivity of BRAF-mutated cells, consistently with the chaperoning activity of HSP90 chaperones toward BRAF. TRAP1 is a HSP90 molecular chaperone deregulated in human tumours and responsible for specific features of cancer cells, i.e., protection from apoptosis, drug resistance, metabolic regulation, and protein quality control/ubiquitination. Because HSP90 is the main molecular chaperone responsible for BRAF folding, with specific affinity greater for its mutated form, HSP90 targeting is presently evaluated as an antitumour strategy in human BRAF-mutated neoplasms. In this scenario, we found that TRAP1 could play a role in radiation resistance. Indeed, transfection of resistant HT29 cells with TRAP1 siRNAs increased cancer cell killing. The role of TRAP1 in radioresistance is confirmed in stable clones of HCT116 cells in which TRAP1 is silenced. Lastly, with daily irradiation the expression of TRAP1 increased, suggesting a role also in the adaptive response to radiation. Conclusions Concurrent radiotherapy and radiation sensitizing 5-FU based chemotherapy is a common treatment strategy for colorectal malignancies. Despite aggressive chemoradiotherapy, a subgroup of tumours which undergo to chemoradiation do not achieve a clinically valuable response and local failure remains a troubling clinical problem that requires development of more effective regimens. In such a context, CRCs are known to frequently have activation of the Ras-Raf-MEK-ERK pathway via activating Ras/Braf mutations or EGFR pathway activation, and this likely represents a mechanism responsible for resistance to chemoradiation. Specifically, our study highlight the relevance of BRAF mutations as determinant of radioresistance. To improve outcomes in patients with CRCs with a BRAF mutation, there is a critical need to better understand the mechanisms of resistance. We describe our attempts to enhance the radiation response with 5-FU. Our data suggest that concurrent treatment with 5-FU may be used in patients with colorectal malignancies to augment radiation response. Our study shows that in CRC cell lines there is a relationship between V600E BRAF mutation and response to radiation with or without 5-FU, suggesting that BRAF mutation might be used as a predictive biomarker of response to neoadjuvant therapy in CRCs. However, the low frequency of V600E BRAF mutation must be considered (3-15%), as well as the need to confirm in vivo the results we have obtained. Our study also suggests that the inhibition of molecules as TRAP1, involved in the regulation of B-Raf, may represent a treatment strategy for V600E-mutated CRCs, a subgroup characterized by a more aggressive biological behavior and a reduced responsiveness to conventional treatments. In conclusion, our study may aid in better understanding the molecular mechanism that control response to radiation in cancer cells. Additionally, our findings may contribute to the development of more effective strategies of combining radiation therapy with other systemic therapies. Our results provide rational therapeutic strategies for clinical studies in this poor prognosis subtype of CRC

Cyclin-dependent kinase 1 targeting improves sensitivity to radiation in BRAF V600E colorectal carcinoma cells

Objectives: Preoperative chemoradiation is currently the standard of care in locally advanced rectal carcinoma, even though a subset of rectal tumors does not achieve major clinically meaningful responses upon neoadjuvant chemoradiation. At present, no molecular biomarkers are available to predict response to neoadjuvant chemoradiation and select resistant tumors willing more intense therapeutic strategies. Thus, BRAF mutational status was investigated for its role in favoring resistance to radiation in colorectal carcinoma cell lines and cyclin-dependent kinase 1 as a target to improve radiosensitivity in BRAF V600E colorectal tumor cells. Methods: Colony-forming assay and apoptotic rates were evaluated to compare the sensitivity of different colon carcinoma cell lines to ionizing radiation and their radiosensitivity upon exposure to BRAF and/or cyclin-dependent kinase 1 inhibitory/silencing strategies. Cyclin-dependent kinase 1 expression/subcellular distribution was studied by immunoblot analysis. Results: Colon carcinoma BRAF V600E HT29 cells exhibited poor response to radiation compared to BRAF wild-type COLO320 and HCT116 cells. Interestingly, neither radiosensitizing doses of 5-fluoruracil nor BRAF inhibition/silencing significantly improved radiosensitivity in HT29 cells. Of note, poor response to radiation correlated with upregulation/relocation of cyclin-dependent kinase 1 in mitochondria. Consistently, cyclin-dependent kinase 1 inhibition/silencing as well as its targeting, through inhibition of HSP90 quality control pathway, significantly inhibited the clonogenic ability and increased apoptotic rates in HT29 cells upon exposure to radiation. Conclusion: These data suggest that BRAF V600E colorectal carcinoma cells are poorly responsive to radiation, and cyclin-dependent kinase 1 represents a target to improve radiosensitivity in BRAF V600E colorectal tumor cells

Supplementary_figure_1 – Supplemental material for Cyclin-dependent kinase 1 targeting improves sensitivity to radiation in BRAF V600E colorectal carcinoma cells

<p>Supplemental material, Supplementary_figure_1 for Cyclin-dependent kinase 1 targeting improves sensitivity to radiation in BRAF V600E colorectal carcinoma cells by Girolamo Spagnoletti, Valeria Li Bergolis, Annamaria Piscazzi, Francesca Giannelli, Valentina Condelli, Lorenza Sisinni, Giuseppe Bove, Giovanni Storto and Matteo Landriscina in Tumor Biology</p

Disease-specific and general health-related quality of life in newly diagnosed prostate cancer patients: The Pros-IT CNR study

BACKGROUND: The National Research Council (CNR) prostate cancer monitoring project in Italy (Pros-IT CNR) is an observational, prospective, ongoing, multicentre study aiming to monitor a sample of Italian males diagnosed as new cases of prostate cancer. The present study aims to present data on the quality of life at time prostate cancer is diagnosed. METHODS: One thousand seven hundred five patients were enrolled. Quality of life is evaluated at the time cancer was diagnosed and at subsequent assessments via the Italian version of the University of California Los Angeles-Prostate Cancer Index (UCLA-PCI) and the Short Form Health Survey (SF-12). RESULTS: At diagnosis, lower scores on the physical component of the SF-12 were associated to older ages, obesity and the presence of 3+ moderate/severe comorbidities. Lower scores on the mental component were associated to younger ages, the presence of 3+ moderate/severe comorbidities and a T-score higher than one. Urinary and bowel functions according to UCLA-PCI were generally good. Almost 5% of the sample reported using at least one safety pad daily to control urinary loss; less than 3% reported moderate/severe problems attributable to bowel functions, and sexual function was a moderate/severe problem for 26.7%. Diabetes, 3+ moderate/severe comorbidities, T2 or T3-T4 categories and a Gleason score of eight or more were significantly associated with lower sexual function scores at diagnosis. CONCLUSIONS: Data collected by the Pros-IT CNR study have clarified the baseline status of newly diagnosed prostate cancer patients. A comprehensive assessment of quality of life will allow to objectively evaluate outcomes of different profile of care

Disease-specific and general health-related quality of life in newly diagnosed prostate cancer patients: the Pros-IT CNR study

Background: The National Research Council (CNR) prostate cancer monitoring project in Italy (Pros-IT CNR) is an observational, prospective, ongoing, multicentre study aiming to monitor a sample of Italian males diagnosed as new cases of prostate cancer. The present study aims to present data on the quality of life at time prostate cancer is diagnosed. Methods: One thousand seven hundred five patients were enrolled. Quality of life is evaluated at the time cancer was diagnosed and at subsequent assessments via the Italian version of the University of California Los Angeles-Prostate Cancer Index (UCLA-PCI) and the Short Form Health Survey (SF-12). Results: At diagnosis, lower scores on the physical component of the SF-12 were associated to older ages, obesity and the presence of 3+ moderate/severe comorbidities. Lower scores on the mental component were associated to younger ages, the presence of 3+ moderate/severe comorbidities and a T-score higher than one. Urinary and bowel functions according to UCLA-PCI were generally good. Almost 5% of the sample reported using at least one safety pad daily to control urinary loss; less than 3% reported moderate/severe problems attributable to bowel functions, and sexual function was a moderate/severe problem for 26.7%. Diabetes, 3+ moderate/severe comorbidities, T2 or T3-T4 categories and a Gleason score of eight or more were significantly associated with lower sexual function scores at diagnosis. Conclusions: Data collected by the Pros-IT CNR study have clarified the baseline status of newly diagnosed prostate cancer patients. A comprehensive assessment of quality of life will allow to objectively evaluate outcomes of different profile of care

Pros-IT CNR: an Italian prostate cancer monitoring project

Aims: The Pros-IT CNR project aims to monitor a sample of Italian males \ue2\u89\ua518\uc2&nbsp;years of age who have been diagnosed in the participating centers with incident prostate cancer, by analyzing their clinical features, treatment protocols and outcome results in relation to quality of life. Methods: Pros-IT CNR is an observational, prospective, multicenter study. The National Research Council (CNR), Neuroscience Institute, Aging Branch (Padua) is the promoting center. Ninety-seven Italian centers located throughout Italy were involved. The field study began in September 1, 2014. Subjects eligible were diagnosed with biopsy-verified prostate cancer, na\uc3\uafve. A sample size of 1500 patients was contemplated. A baseline assessment including anamnestic data, clinical history, risk factors, the initial diagnosis, cancer staging information and quality of life (Italian UCLA Prostate Cancer Index; SF-12 Scale) was completed. Six months after the initial diagnosis, a second assessment evaluating the patient\ue2\u80\u99s health status, the treatment carried out, and the quality of life will be made. A third assessment, evaluating the treatment follow-up and the quality of life, will be made 12\uc2&nbsp;months after the initial diagnosis. The 4th, 5th, 6th and 7th assessments, similar to the third, will be completed 24, 36, 48 and 60\uc2&nbsp;months after the initial diagnosis, respectively, and will include also a Food Frequency Questionnaire and the Physical Activity Scale for the Elderly. Discussion: The study will provide information on patients\ue2\u80\u99 quality of life and its variations over time in relation to the treatments received for the prostate cancer