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Upregulation of Aurora kinases has been associated with increased tumor progression, and thus they are appealing targets for the development of anti-cancer therapies. In addition to have a critical role in cell cycle regulation, Aurora kinases have been shown to stabilize MYC-family oncoproteins for the maintenance of malignancy. Aurora kinase inhibitors such as Alisertib has demonstrated compelling anti-tumor efficacies; however, the reported side effects including serious haematological disturbances have limited its risk-benefit ratio in clinical use. In this study we discovered a novel pyrimidine-based Aurora kinase inhibitor compound A that degraded MYC- and MYCN- oncoproteins with better potency than Alisertib. The acyl-based prodrug design leads to the discovery of compound B which was able to regress MYC- or MYCN- overexpressing tumor xenografts including small cell lung cancer, neuroblastoma, hepatocellular carcinoma and medulloblastoma using a once-a-week (QW) oral dosing regimen. Pharmacokinetic studies revealed that the tumor/plasma ratio of compound B was about 20 at 24 h post drug administration, and the active compound remained detectable in the tumors after 7 days. No significant haematological or liver/kidney biochemical aberration was observed in mice treated with up to 500 mg/kg of DBPR728 on a QW dosing regimen in a 21-day cycle. The unique pharmacokinetic and molecular properties of compound B hold potential clinical promise for treating MYC- and MYCN- amplified tumors with manageable on-target haematological adverse effects caused by Aurora kinase inhibition. Citation Format: Ya-Hui Chi, Chun-Ping Chang, Teng-Kuang Yeh, Wan-Ping Wang, Yen-Ting Chen, Chia-Hua Tsai, Yan-Fu Chen, Yi-Yu Ke, Jing-Ya Wang, Ching-Ping Chen, Tsung-Chih Hsieh, Mine-Hsine Wu, Chiung-Tong Chen. Targeting myc-amplified cancers with a novel prodrug inhibiting aurora A kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 523.

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. How DNA-PK is activated in response to DSBs has remained elusive. Here, we show that the SIRT2 sirtuin deacetylase and tumor suppressor directs the activation of DNA-PK through deacetylation of its catalytic subunit (DNA-PKcs). SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR, which facilitates its interaction with Ku and recruitment to DSBs, thereby leading to DNA-PKcs autophosphorylation and DNA-PK signaling to downstream NHEJ substrates. Moreover, SIRT2 inhibitor sensitizes resistant cancer cells and tumors to IR. Our findings define a mechanism for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ to promote genome integrity and govern IR resistance, which can be exploited for improvements in cancer therapy. Citation Format: PamelaSara E. Head, Nagaraju P. Ganji, Shi-Ya Wang, Duc Duong, Hui Zhang, Waaqo Daddacha, Shuyi Li, Nicholas T. Seyfried, David M. Smalley, Ya Wang, Xingming Deng, William S. Dynan, Bassel El-Rayes, Anthony J. Davis, David S. Yu. DNA-PKCS deacetylation by SIRT2 promotes DNA double-strand break repair by non-homologous end joining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2561.

Loss or attenuated expression of the tumor-suppressor gene FHIT is associated paradoxically with poor progression of human tumors. Fhit promotes apoptosis and regulates reactive oxygen species; however, the mechanism by which Fhit inhibits tumor growth in animals remains unclear. In this study, we used a multidisciplinary approach based on bioinformatics, small RNA library screening, human tissue analysis, and a xenograft mouse model to identify a novel member of the miR-548 family in the fourth intron of the human FHIT gene. Characterization of this human-specific microRNA illustrates the importance of this class of microRNAs in tumor suppression and may influence interpretation of Fhit action in human cancer. Cancer Res; 74(8); 2283–94. ©2014 AACR.

MicroRNA-21 (miR-21) is an oncomir overexpressed in most human tumors in that it promotes malignant growth and progression by acting on multiple targets. Here, we broaden the impact of miR-21 in cancer by showing that it regulates the formation of reactive oxygen species (ROS) that promote tumorigenesis. Key targets of miR-21 in mediating this function were SOD3 and TNFα. We found that miR-21 inhibited the metabolism of superoxide to hydrogen peroxide, produced either by endogenous basal activities or exposure to ionizing radiation (IR), by directing attenuating SOD3 or by an indirect mechanism that limited TNFa production, thereby reducing SOD2 levels. Importantly, both effects contributed to an elevation of IR-induced cell transformation. Our findings, therefore, establish that miR-21 promotes tumorigenesis to a large extent through its regulation of cellular ROS levels. Cancer Res; 72(18); 4707–13. ©2012 AACR.

The glutathione (GSH) overexpression small-cell-lung-cancer cell lines, SR3A-13 and SR3A-14, transfected with recombinant DNA encoding γ-glutamylcysteine synthetase (γ-GCSh), are radiation resistant. However, they are more sensitive to cisplatin treatment, compared with parental cell (SR3A)1. In addition to be a copper transporter, the hCtr-1 is also a transporter for platinum (Pt)-based agents. Therefore, the cellular uptake of cisplatin is enhanced by the upregulation of human copper transporter 1 (hCtr-1) at the cell membrane. We have synthesized an alloy FePt nanoparticles (FePt NPs) that generated excellent contrast effects in both CT and MRI images2. Interestingly, the uptake of FePt NPs by SR3A-13 and -14 cells was 3-3.5 fold higher than that of SR3A cells, measured with inductively coupled plasma optical emission spectrometry (ICP-OES) and transmission electron microscopy (TEM). Because intracellular NPs have been shown to generate reactive oxygen species (ROS) and overproduction of ROS may enhance the efficacy of radiotherapy, we sought to explore whether treatment with the alloy FePt NPs can reverse the radiation resistance in SR3A-13 and -14 cells. We found co-treatment with the alloy FePt NPs and radiation effectively reduce the survivals of these 2 cells. Furthermore, we found an alternation in mitochondrial appearance accompanied with low signal of Tetramethylrhodamine, methyl ester (TMRM), indicating a malfunction is induced in mitochondria. We conclude that FePt NPs are able to overcome radiation resistance in hCtr-1 high expression cells via mitochondria targeting. Citation Format: Tsung-Lin Tsai, Macus Tien Kuo, Jen-Ya Wang, Wan-Li Shin, Helen H. W. Chen, Wu-Chou Su. FePt nanoparticles overcoming radiotherapy resistance in hCtr-1 high expression cells via mitochondria targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3214.

Fragile histidine triad (FHIT) gene deletion or promoter methylation and reduced Fhit protein expression occur in ∼70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified. We previously reported that compared with Fhit+/+ cells, Fhit−/− cells with an overactivated ATR/CHK1 pathway show increased mutation frequency and resistance to DNA damage–induced killing, indicating that Fhit and the CHK1 pathway have opposing roles in cells responding to DNA damage. In this study, we show that cells, with or without Fhit expression, have similar DNA double-strand break induction levels and similar rejoining rates following ionizing radiation, indicating that the effect of Fhit on cell radiosensitivity is independent of nonhomologous end-joining. By combining I-SceI–induced-DNA double-strand break system and small interfering RNA approach, we also show that knocking down Fhit increases the efficiency of homologous recombination repair of cells, but knocking down Chk1 decreases the efficiency of homologous recombination repair, associated with the sensitivity to ionizing radiation–induced killing. Taken together, the results show that the role of Fhit in affecting the sensitivity of cells to ionizing radiation–induced killing is through the CHK1 pathway linked to homologous recombination repair. These results also illustrate the importance of balanced checkpoint activation in genomic stability and suggest a connection between the radioresistance and mutagenesis, carcinogenesis, as well as tumor progression in Fhit-deficient cells or tissue.

Human tumor cell death during radiotherapy is caused mainly by ionizing radiation (IR)–induced DNA double-strand breaks (DSB), which are repaired by either homologous recombination repair (HRR) or nonhomologous end-joining (NHEJ). Although siRNA-mediated knockdown of DNA DSB repair genes can sensitize tumor cells to IR, this approach is limited by inefficiencies of gene silencing. In this study, we show that combining an artificial miRNA (amiR) engineered to target 3′-untranslated regions of XRCC2 (an HRR factor) or XRCC4 (an NHEJ factor) along with an siRNA to target the gene coding region can improve silencing efficiencies to achieve more robust radiosensitization than a single approach alone. Mechanistically, the combinatorial knockdown decreased targeted gene expression through both a reduction in mRNA stability and a blockade to mRNA translation. Together, our findings establish a general method of gene silencing that is more efficient and particularly suited for suppressing genes that are difficult to downregulate by amiR- or siRNA-based methods alone. Cancer Res; 72(5); 1221–8. ©2012 AACR.

Understanding the molecular details associated with aberrant high mobility group A2 (HMGA2) gene expression is key to establishing the mechanism(s) underlying its oncogenic potential and effect on the development of therapeutic strategies. Here, we report the involvement of HMGA2 in impairing DNA-dependent protein kinase (DNA-PK) during the nonhomologous end joining (NHEJ) process. We showed that HMGA2-expressing cells displayed deficiency in overall and precise DNA end-joining repair and accumulated more endogenous DNA damage. Proper and timely activation of DNA-PK, consisting of Ku70, Ku80, and DNA-PKcs subunits, is essential for the repair of DNA double strand breaks (DSB) generated endogenously or by exposure to genotoxins. In cells overexpressing HMGA2, accumulation of histone 2A variant X phosphorylation at Ser-139 (γ-H2AX) was associated with hyperphosphorylation of DNA-PKcs at Thr-2609 and Ser-2056 before and after the induction of DSBs. Also, the steady-state complex of Ku and DNA ends was altered by HMGA2. Microirradiation and real-time imaging in living cells revealed that HMGA2 delayed the release of DNA-PKcs from DSB sites, similar to observations found in DNA-PKcs mutants. Moreover, HMGA2 alone was sufficient to induce chromosomal aberrations, a hallmark of deficiency in NHEJ-mediated DNA repair. In summary, a novel role for HMGA2 to interfere with NHEJ processes was uncovered, implicating HMGA2 in the promotion of genome instability and tumorigenesis. [Cancer Res 2009;69(14):5699–706]

ATR is one of the most important checkpoint proteins in mammalian cells responding to DNA damage. Cells defective in normal ATR activity are sensitive to ionizing radiation (IR). The mechanism by which ATR protects the cells from IR-induced killing remains unclear. DNA double-strand breaks (DSBs) induced by IR are critical lesions for cell survival. Two major DNA DSB repair pathways exist in mammalian cells: homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). We show that the doxycycline (dox)-induced ATR kinase dead (ATRkd) cells have the similar inductions and rejoining rates of DNA DSBs compared with cells without dox induction, although the dox-induced ATRkd cells are more sensitive to IR and have the deficient S and G2 checkpoints. We also show that the dox-induced ATRkd cells have a lower HRR efficiency compared with the cells without dox induction. These results indicate that the effects of ATR on cell radiosensitivity are independent of NHEJ but are linked to HRR that may be affected by the deficient S and G2 checkpoints.

There are two major pathways for repairing DNA double strand breaks in mammalian cells: nonhomologous end joining (NHEJ) and homologous recombination repair (HRR). The nonhomologous end joining repair is deficient in cells without Ku, whereas HRR is highly efficient in such cells compared with their wild-type counterparts. The mechanism remains unclear. We reported previously that Ku80(-/-) cells show a stronger ATM-dependent S-phase checkpoint response than Ku80(+/+) cells after ionizing radiation (IR; X-Y. Zhou et al., Oncogene, 21:6377-6381, 2002). We report in this study that Ku80(-/-) cells also show a much stronger G(2) accumulation than Ku80(+/+) cells after IR. The stronger G(2) checkpoint response in Ku80(-/-) cells is ATM independent but is accompanied with a higher activity of CHK1 kinase. Treatment with Chk1 antisense oligonucleotide abolishes the stronger G(2) checkpoint response and sensitizes Ku80(-/-) cells to IR. These data indicate that the stronger G(2) checkpoint response shown in Ku80(-/-) cells is CHK1 dependent and suggest that the CHK1-dependent checkpoint response contributes to the highly efficient HRR in such cells.

Camptothecin (CPT) that targets DNA topoisomerase I is one of the most promising broad-spectrum anticancer drugs in development today. The cytotoxicity of CPT is S phase (S)-specific because the collision of advancing replication forks with CPT-topoisomerase I-DNA complexes results in DNA damage. After DNA damage, proliferating cells could actively slow down the DNA replication through an S checkpoint to provide time for repair. We report now that there is an activated S checkpoint response in CPT-treated mammalian cells. This response is regulated by Ataxia and Rad3-related (ATR)/CHK1 pathway. Compared with their wild-type counterparts, CPT-treated Ku80-/- cells showed stronger inhibition of DNA replication. This stronger inhibition had no relationship with DNA-dependent protein kinase (DNA-PK) activity but correlated with the higher activities of ATR and the higher activities of CHK1 in such cells. Not only caffeine, the nonspecific inhibitor of ATR, or UCN-01, the nonspecific inhibitor of CHK1, but also the specific CHK1 antisense oligonucleotide abolished the stronger inhibition of DNA replication in CPT-treated Ku80-/- cells. These results in aggregate indicated that the stronger S checkpoint in CPT-treated Ku80-/- cells is regulated through the highly activated ATR/CHK1 pathway.

Background: c-Met is a receptor tyrosine kinase (RTK) that has been shown to be a potential target of therapy in a variety of human cancers. We investigated if c-Met served as a potential therapeutic target in esophageal squamous cell cancer (ESCC). Methods: Cytotoxicity (MTT) assay, cell migration assay and cell cycle analysis by flow cytometry were conducted in KYSE 510 and 170 ESCC cell lines (kindly provided by Dr. Yutaka Shimada) in response to various concentrations of c-met inhibitors, SU11274 and PHA665752. Apoptosis of ESCC cell lines was detected by TUNEL assay. Cell cycle regulatory proteins, cyclin A and B1, tumor suppressors including Rb, p53 p21 and p27, and intrinsic apoptotic factors were determined by Western blot analysis. Mice inoculated with KYSE 170 ESCC cell line were used to test therapeutic response of c-met inhibitors in vivo. Results: SU11274 and PHA665752 significantly suppressed viability and migration of KYSE 170 and 510 ESCC cells with decreased expression of phosphorylated c-Met (p-c-Met). Expression of p21, p27, p53 and Rb was upregulated in ESCC cell lines treated with c-Met inhibitors while inducing G1/G2 cell cycle arrest and apoptosis. Tumor growth in vivo was significantly suppressed in response to treatment with c-Met inhibitors with decreased expression of p-c-Met within the mice tumors. Conclusion: c-Met inhibitors exerted significant anticancer effects in vitro and in vivo for ESCC cell lines, suggesting that c-Met may serve as a potential therapeutic target for ESCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2878. doi:1538-7445.AM2012-2878

During radiotherapy, human tumor cell death is mainly caused by ionizing radiation (IR)-induced DNA double strand breaks (DSBs). In general, human cells use two pathways: homologous recombination repair (HRR) and non-homologous end-joining (NHEJ), to repair DNA DSBs. However, in many cases, human tumors are resistant to IR due to the abnormal up-regulation of DNA DSB repair genes. Previously it was reported that using a siRNA approach could knock down the DNA DSB repair genes and sensitized the tumor cells to IR, however, the levels of knock down using a siRNA approach alone is limited. Here, we developed a novel combined approach using an artificial microRNA (amiR) to target the 3′-untranlated region (UTR) of the DNA DSB repair gene, XRCC2 (a HRR factor) or XRCC4 (a NHEJ factor), and the siRNA to target the coding region of the gene in different human tumor cell lines. Our results indicate that the combined approach is much more efficient than the amiR or siRNA approach alone in knocking down the targeted gene and sensitizing human tumor cells to IR-induced killing. The amiR decreased the targeted gene through reducing the mRNA stability and blocking the translation. These results demonstrate that the combined approach could be used for many different purposes to knock down genes in different cell lines, especially genes that are difficult to knock down using amiR or siRNA alone. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1106. doi:1538-7445.AM2012-1106

Background: Camptothecin (CPT), an inhibitor of DNA topoisomerase I, induces replication related DNA double strand breaks (DSBs). Previously, it was reported that Ku-dependent non-homologous end-joining (NHEJ) deficient DT40 cells were more resistant to CPT-induced killing than wild type cells (JBC, 279:37343, 2004), however, the mechanism remains unclear. We were interested in testing the hypothesis that Mre11 binding to DNA double strand break (DSB) (the first step for initiating homologous recombination repair (HRR)) is interfered by the Ku-dependent NHEJ and the interference was much stronger in S phase. Methods: Different fork-related DNA structures were designed for testing the efficiency of purified Mre11 protein (kindly provided by Dr. Tainer) in vitro. DT40 cell lines: wild type, Ku70-/, DNA-PKcs-/-, Rad54-/- and Rad51 conditional knock out cells were used in this study. The efficiency Mre11 binding to chromatin DNA in CPT treated the cell lines were detected. The cell cycle distribution was measured by a flow cytometer. The sensitivity of these cell lines to CPT were measured by a clonogenic assay. Results: Compared with their wild-type counterparts, Rad54−/− and Rad51 conditional knock out cells (essential factors for (HRR)) were much more sensitive to CPT-induced killing but Ku70−/− and DNA-PKcs cells (essential factors for non-homologous end joining (NHEJ)) were much more resistant to CPT-treated killing, which was accompanied by a huge S-phase block in these resistant cells. Mre11 bound to DNA substrates especially to the mimic DNA replication fork substrates. Mre11 bound to chromatin DNA in Ku70-/- and DNA-PKcs-/- cells stronger than that in wild type cells following CPT treatment. Conclusion: CPT-induced S checkpoint facilitates Mre11 binding and promotes HRR, which protects cells from CPT-induced killing. The Ku-dependent NHEJ block efficiency of Mre11 binding to DNA substrates especially DNA replication related structure, which inhibits HRR and renders sensitivity of the cells to CPT treatment. 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 2520. doi:10.1158/1538-7445.AM2011-2520

Background: Previously, we reported that MEPE/OF45 interacting with CHK1 protects CHK1 from degradation and cells from DNA damage-induced killing (Nucleic Acids Res, 37: 7447-7454 2009; Brit J Cancer 102: 862-866, 2010). To further investigate the nature of the CHK1 stabilization and its effects on cell sensitivity to DNA damage inducers, we designed this study to identify the key domain of CHK1 interaction with MEPE/OF45 and the key ubiquitination site of CHK1. Methods: GST-MEPE/OF45 was expressed in E. coli BL 21. HA-wild type CHK1 and various CHK1 truncated mutations were expressed in 293FT. GST pull-down assay was used to identify the key domain of CHK1 interacting with MEPE/OF45. To identify the ubiquitination site of CHK1, GFP-CHK1 with different Lys mutations were co-transfected with Ub-expression vector to 293FT, and 48 hours later, whole cell lysate were prepared, Western blot was carried out using a GFP monoclonal antibody to detect ubiquitinaed CHK1. The CHK1 half-life was used to detect the stabilization of CHK1 and a clonogenic assay was used to detect the sensitivity of cells to DNA damage inducers. Results: The key domain of CHK1 interacting with MEPE/OF45 and the key site of CHK1 ubiquitination were identified. Most importantly, the mutation of CHK1 with the main domain or key site affected CHK1 stabilization and cell sensitivity to DNA damage inducers. Conclusion: The interaction of CHK1 with MEPE/OF45 plays an important role in preventing CHK1 ubiquitination and; therefore, stabilizes CHK1, which further affects cell sensitivity to DNA damage inducers. Because CHK1 is an essential DNA damage response protein, these results provide valuable information for understanding the role of CHK1 in genomic integrity, which could be used for improving cancer treatment. 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 4059. doi:10.1158/1538-7445.AM2011-4059

Background: Previously, we reported that targeting DNA repair genes with miR-101 could sensitize human brain tumor cells to radiation (PLoS One 5: e11397, 2010). However, some tumor cell lines with high levels of endogenous miR-101 showed less response to exogenous miR-101 (our unpublished data). To overcome the resistance of the tumors to radiation, we designed artificial miRNAs to target XRCC2 (an important factor for homologous recombination repair (HRR)) and XRCC4 (an essential factor for non-homologous end-joining (NHEJ)) because both HRR and NHEJ are required for repairing radiation-induced DNA double strand breaks (DSBs) in mammalian cells and DNA DSBs are the severest threat to cell survival. Methods: We constructed pLenti6/V5-GW/Em-GFP-miR lentiviral expression plasmids expressing artificial XRCC4 and XRCC2 miRNAs using Gateway Technology and chained artificial XRCC4 and XRCC2 miRNAs. To avoid triggering miRNA tailing and 3′-to-5′ trimming due to an extensive complementarity between a target RNA and an Argonaute1-bound miRNA, we mismatched 7 bases in the central bulge of the artificial miRNA. U87MG cells were transfected with the vector encoding both of the artificial miRNAs. XRCC4 and XRCC2 levels were measured by real-time PCR and Western blot. The effects of artificial miRNA on sensitizing U87MG cells to radiation were determined by clonogenic assay. Results: The designed artificial XRCC4/XRCC2-miRNA could be over-expressed in U87MG cells and efficiently down-regulate the XRCC4 and XRCC2 levels. Most importantly, the artificial XRCC4/XRCC2-miRNA could efficiently sensitize the brain tumor cells to radiation. Conclusion: The designed artificial miRNA targeting XRCC4 and XRCC2 could be a good tool to sensitize brain tumor cells to radiation, which provide a new approach for potentially improving radiotherapy. 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 148. doi:10.1158/1538-7445.AM2011-148

Radiotherapy is a common treatment strategy for many solid tumors, including breast cancer. Unfortunately, solid tumors can be resistant or develop resistance to radiotherapy. Though many studies have been conducted to further elucidate the mechanisms of radioresistance, they remain largely obscure due to the complex genetic cellular response to radiation and involvement of a large number of genes. MicroRNA, a recently discovered class of small, functional, non-coding RNA, is one of such class of potential regulators. A single microRNA can target a large number of genes and regulate several signal pathways simultaneously. Thus, miRNAs may be involved in the complex radioresistance of tumor cells. In the present study, we characterized miR-302 family members (miR-302s) pre-clinically as novel biomarkers for radiotherapy resistance. Our results showed that miR-302s were downregulated in irradiated breast cancer cells. Furthermore, we found, with a bioinformatic analysis, that AKT and RAD52, two critical regulators of radioresistance, are the predicted targets of miR-302s. Additionally, the expression levels of miR-302a were inversely consistently with those of AKT and RAD52. More promisingly, our data have demonstrated that miR-302a mimics sensitized breast cancer MDA-MB-231 cells to radiation therapy and reduced the expression of AKT and RAD52. Collectively, loss of miR-302s may be involved in the radioresistance of breast cancer cells, mainly through the release of repression of AKT and RAD52. Thus, our findings suggest that decreased expression of miR-302s contributes to radiotherapy-resistance and restoration of miR-302 baseline expression sensitizes breast cancer cells to radiotherapy. These data suggest that miR-302s may be potential sensitizers to radiotherapy and represent a novel strategy for radiotherapy of breast cancer. 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 2670. doi:10.1158/1538-7445.AM2011-2670