Edward Graves, Postdoctoral Faculty Sponsor
Long non-coding RNAs (lncRNAs) have been shown to impact important biological functions such as proliferation, survival, and genomic stability. To analyze radiation-induced lncRNA expression in human tumors, we irradiated prostate cancer cells with a single dose of 10Gy or a multifractionated radiotherapeutic regimen of 10 fractions with a dose of 1Gy (10* 1 Gy) during 5days. We found a stable upregulation of the lncRNA LINC00261 and LINC00665 at 2months after radiotherapy that was more pronounced after single-dose irradiation. Analysis of the The Cancer Genome Atlas (TCGA) and The Atlas of Non-coding RNAs in Cancer (TANRIC) databases showed that high expression of these two lncRNAs may be a potential negative prognostic marker for overall survival of prostate cancer patients. Knockdown of LINC00261 and LINC00665 in long-term survivors decreased survival after re-irradiation and affected DNA double-strand break repair. Mechanistically, both lncRNAs showed an interdependent expression and regulated expression of the DNA repair proteins CtIP (RBBP8) and XPC as well as the microRNA miR-329. Identifying long-term tumor adaptation mechanisms can lead to the discovery of new molecular targets, in effect opening up new research directions and improving multimodal radiation oncologic treatment.
View details for DOI 10.1016/j.omtn.2021.02.024
View details for PubMedID 33767914
Radiation therapy (RT) is highly effective due to its ability to physically focus the treatment to target the tumor while sparing normal tissue and its ability to be combined with systemic therapy. This systemic therapy can be utilized before RT as an adjuvant or induction treatment, during RT as a radiation "sensitizer," or following RT as a part of combined modality therapy. As part of a unique concept of using radiation as "focused biology" we investigated how tumors and normal tissues adapt to clinically relevant multi-fraction (MF) and single-dose (SD) radiation to observe whether the adaptations can induce susceptibility to cell killing by available drugs or by immune enhancement. We identified an adaptation occurring after MF (3 x 2 Gy) that induced cell killing when AKT-mTOR inhibitors were delivered following cessation of RT. Additionally, we identified inducible changes in integrin expression 2 months following cessation of RT that differ between MF (1 Gy x 10) and SD (10 Gy) that remain targetable compared to pre-RT. Adaptation is reflected across different "omics" studies, and thus the range of possible molecular targets is not only broad but also time, dose, and schedule dependent. While much remains to be studied about the radiation adaptive response, radiation should be characterized by its molecular perturbations in addition to physical dose. Consideration of the adaptive effects should result in the design of a tailored radiotherapy treatment plan that accounts for specific molecular changes to be targeted as part of precision multi-modality cancer treatment.
View details for DOI 10.1158/1078-0432.CCR-20-0572
View details for PubMedID 32554542
Multifractionated irradiation is the mainstay of radiation treatment in cancer therapy. Yet, little is known about the cellular DNA repair processes that take place between radiation fractions, even though understanding the molecular mechanisms promoting cancer cell recovery and survival could improve patient outcome and identify new avenues for targeted intervention. To address this knowledge gap, we systematically characterized how cells respond differentially to multifractionated and single-dose radiotherapy, using a combination of genetics-based and functional approaches. We found that both cancer cells and normal fibroblasts exhibited enhanced survival after multifractionated irradiation compared with an equivalent single dose of irradiation, and this effect was entirely dependent on 53BP1-mediated NHEJ. Furthermore, we identified RIF1 as the critical effector of 53BP1. Inhibiting 53BP1 recruitment to damaged chromatin completely abolished the survival advantage after multifractionated irradiation and could not be reversed by suppressing excessive end resection. Analysis of the TCGA database revealed lower expression of 53BP1 pathway genes in prostate cancer, suggesting that multifractionated radiotherapy might be a favorable option for radio-oncologic treatment in this tumor type. We propose that elucidation of DNA repair mechanisms elicited by different irradiation dosing regimens could improve radiotherapy selection for the individual patient and maximize the efficacy of radiotherapy.
View details for DOI 10.1093/nar/gkz1139
View details for PubMedID 31822909
Innovation and progress in radiation oncology depend on discovery and insights realized through research in radiation biology. Radiobiology research has led to fundamental scientific insights, from the discovery of stem/progenitor cells to the definition of signal transduction pathways activated by ionizing radiation that are now recognized as integral to the DNA damage response (DDR). Radiobiological discoveries are guiding clinical trials that test radiation therapy combined with inhibitors of the DDR kinases DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), ataxia telangiectasia related (ATR), and immune or cell cycle checkpoint inhibitors. To maintain scientific and clinical relevance, the field of radiation biology must overcome challenges in research workforce, training, and funding. The National Cancer Institute convened a workshop to discuss the role of radiobiology research and radiation biologists in the future scientific enterprise. Here, we review the discussions of current radiation oncology research approaches and areas of scientific focus considered important for rapid progress in radiation sciences and the continued contribution of radiobiology to radiation oncology and the broader biomedical research community.
View details for PubMedID 29126306
View details for PubMedCentralID PMC5928778
Adaptation of tumor cells to radiotherapy induces changes that are actionable by molecular targeted agents and immunotherapy. This report demonstrates that radiation-induced changes in integrin expression can be targeted 2 months later. Integrins are transmembrane cell adhesion molecules that are essential for cancer cell survival and proliferation. To analyze the short- and long-term effects of radiation on the integrin expression, prostate cancer cells (DU145, PC3, and LNCaP) were cultured in a 3D extracellular matrix and irradiated with either a single dose of radiation (2-10 Gy) or a multifractionated regimen (2-10 fractions of 1 Gy). Whole human genome microarrays, immunoblotting, immunoprecipitation assays, and immunofluorescence staining of integrins were performed. The results were confirmed in a prostate cancer xenograft model system. Interestingly, ?1 and ?4 integrins (ITGB1 and ITGB4) were upregulated after radiation in vitro and in vivo. This overexpression lasted for more than 2 months and was dose dependent. Moreover, radiation-induced upregulation of ?1 and ?4 integrin resulted in significantly increased tumor cell death after treatment with inhibitory antibodies. Combined, these findings indicate that long-term tumor adaptation to radiation can result in an increased susceptibility of surviving cancer cells to molecular targeted therapy due to a radiation-induced overexpression of the target. IMPLICATIONS: Radiation induces dose- and schedule-dependent adaptive changes that are targetable for an extended time; thus suggesting radiotherapy as a unique strategy to orchestrate molecular processes, thereby providing new radiation-drug treatment options within precision cancer medicine.
View details for DOI 10.1158/1541-7786.MCR-18-0232
View details for PubMedID 30042176
View details for PubMedCentralID PMC6279542
Implementing targeted drug therapy in radio-oncologic treatment regimens has greatly improved the outcome of cancer patients. However, the efficacy of molecular targeted drugs such as inhibitory antibodies or small molecule inhibitors essentially depends on target expression and activity, which both can change during the course of treatment. Radiotherapy has previously been shown to activate prosurvival pathways, which can help tumor cells to adapt and thereby survive treatment. Therefore, we aimed to identify changes in signaling induced by radiation and evaluate the potential of targeting these changes with small molecules to increase the therapeutic efficacy on cancer cell survival. Analysis of "The Cancer Genome Atlas" database disclosed a significant overexpression of AKT1, AKT2, and MTOR genes in human prostate cancer samples compared with normal prostate gland tissue. Multifractionated radiation of three-dimensional-cultured prostate cancer cell lines with a dose of 2 Gy/day as a clinically relevant schedule resulted in an increased protein phosphorylation and enhanced protein-protein interaction between AKT and mTOR, whereas gene expression of AKT, MTOR, and related kinases was not altered by radiation. Similar results were found in a xenograft model of prostate cancer. Pharmacologic inhibition of mTOR/AKT signaling after activation by multifractionated radiation was more effective than treatment prior to radiotherapy. Taken together, our findings provide a proof-of-concept that targeting signaling molecules after activation by radiotherapy may be a novel and promising treatment strategy for cancers treated with multifractionated radiation regimens such as prostate cancer to increase the sensitivity of tumor cells to molecular targeted drugs. Mol Cancer Ther; 17(2); 355-67. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."
View details for DOI 10.1158/1535-7163.MCT-17-0262
View details for PubMedID 28802252
View details for PubMedCentralID PMC5805592
Signaling from integrins and receptor tyrosine kinases (RTKs) contributes substantially to therapy resistance of malignant tumors. We investigated simultaneous ?1 integrin-epidermal growth factor receptor (EGFR) targeting plus radiotherapy in human head and neck squamous cell carcinomas (HNSCCs).Ten HNSCC cell lines were grown in three-dimensional laminin-rich extracellular matrix cell cultures and two of them as tumor xenografts in nude mice (n = 12-16 per group). Targeting of ?1 integrin and EGFR with monoclonal inhibitory antibodies (AIIB2 and cetuximab, respectively) was combined with x-ray irradiation. Clonogenic survival, tumor growth, and tumor control (evaluated by Kaplan-Meier analysis), apoptosis, phosphoproteome (interactome, network betweeness centrality analysis), receptor expression (immunohistochemistry), and downstream signaling (western blotting) were assessed. Various mutants of the integrin signaling mediator focal adhesion kinase (FAK) were employed for mechanistic studies. All statistical tests were two-sided.Compared with ?1 integrin or EGFR single inhibition, combined ?1 integrin-EGFR targeting resulted in enhanced cytotoxicity and radiosensitization in eight out of 10 tested HNSCC cell lines, which responded with an FAK dephosphorylation after ?1 integrin inhibition. In vivo, simultaneous anti-?1 integrin/anti-EGFR treatment and radiotherapy of UTSCC15 responder xenografts enabled better tumor control compared with anti-EGFR monotherapy and irradiation (hazard ratio [HR] = 6.9, 95% confidence interval [CI] = 1.6 to 30.9, P = .01), in contrast to the SAS nonresponder tumor model (HR = 0.9, 95% CI = 0.4 to 2.3, P = .83). Mechanistically, a protein complex consisting of FAK- and Erk1-mediated prosurvival signals for radiation resistance, which was effectively compromised by ?1 integrin and EGFR blocking.Concomitant targeting of ?1 integrin and EGFR seems a powerful and promising approach to overcome radioresistance of HNSCCs.
View details for DOI 10.1093/jnci/dju419
View details for PubMedID 25663685
Inherent and acquired resistance to targeted therapeutics continues to emerge as a major clinical obstacle. For example, resistance to EGF receptor targeting occurs commonly, more so than was expected, on the basis of preclinical work. Given emerging evidence that cancer cell-substrate interactions are important determinants of therapeutic sensitivity, we examined the impact of cell-fibronectin interactions on the efficacy of the EGF receptor antibody cetuximab, which is used widely for lung cancer treatment. Our results revealed the potential for cell-fibronectin interactions to induce radioresistance of human non-small cell lung cancer cells. Cell adhesion to fibronectin enhanced tumor cell radioresistance and attenuated the cytotoxic and radiosensitizing effects of cetuximab. Both in vitro and in vivo, we found that cetuximab treatment led to a remarkable induction of fibronectin biosynthesis. Mechanistic analyses revealed the induction was mediated by a p38-MAPK-ATF2 signaling pathway and that RNAi-mediated inhibition of fibronectin could elevate the cytotoxic and radiosensitizing potential of cetuximab. Taken together, our findings show how cell adhesion blunts cetuximab, which, by inducing fibronectin, generates a self-attenuating mechanism of drug resistance.
View details for DOI 10.1158/0008-5472.CAN-13-0344
View details for PubMedID 23950208
EGF receptor (EGFR) promotes tumor growth as well as radio- and chemoresistance in various human malignancies including squamous cell carcinomas (SCC). In addition to deactivation of prosurvival signaling, cetuximab-mediated EGFR targeting might concomitantly induce self-attenuating signaling bypasses. Identification of such bypass mechanisms is key to improve the efficacy of targeted approaches. Here, we show great similarity of EGFR signaling and radiation survival in cetuximab-treated SCC cells grown in a more physiologic three-dimensional extracellular matrix and as tumor xenografts in contrast to conventional monolayer cell cultures. Using phosphoproteome arrays, we observed strong induction of JNK2 phosphorylation potentially resulting from cetuximab-inhibited EGFR through c-jun-NH(2)-kinase (JNK)-interacting protein-4 (JIP-4), which was identified using an immunoprecipitation-mass spectrometric approach. Inhibition of this signaling bypass by JIP-4 or JNK2 knockdown or pharmacologic JNK2 inhibition enhanced cetuximab efficacy and tumor cell radiosensitivity. Our findings add new facets to EGFR signaling and indicate signaling bypass possibilities of cancer cells to improve their survival on cetuximab treatment. By deactivation of cetuximab-self-attenuating JNK2-dependent signaling, the cytotoxicity, and radiosensitizing potential of cetuximab can be augmented.
View details for DOI 10.1158/0008-5472.CAN-12-2021
View details for PubMedID 23074283
Integrin signaling critically contributes to the progression, growth, and therapy resistance of malignant tumors. Here, we show that targeting of ?? integrins with inhibitory antibodies enhances the sensitivity to ionizing radiation and delays the growth of human head and neck squamous cell carcinoma cell lines in 3D cell culture and in xenografted mice. Mechanistically, dephosphorylation of focal adhesion kinase (FAK) upon inhibition of ?? integrin resulted in dissociation of a FAK/cortactin protein complex. This, in turn, downregulated JNK signaling and induced cell rounding, leading to radiosensitization. Thus, these findings suggest that robust and selective pharmacological targeting of ?? integrins may provide therapeutic benefit to overcome tumor cell resistance to radiotherapy.
View details for DOI 10.1172/JCI61350
View details for PubMedID 22378044
View details for PubMedCentralID PMC3314473
Tumor cell resistance to ionizing radiation and chemotherapy is a major obstacle in cancer therapy. One factor contributing to this is integrin-mediated adhesion to ECM. The adapter protein particularly interesting new cysteine-histidine-rich 1 (PINCH1) is recruited to integrin adhesion sites and promotes cell survival, but the mechanisms underlying this effect are not well understood. Here we have shown that PINCH1 is expressed at elevated levels in human tumors of diverse origins relative to normal tissue. Furthermore, PINCH1 promoted cell survival upon treatment with ionizing radiation in vitro and in vivo by perpetuating Akt1 phosphorylation and activity. Mechanistically, PINCH1 was found to directly bind to protein phosphatase 1alpha (PP1alpha) - an Akt1-regulating protein - and inhibit PP1alpha activity, resulting in increased Akt1 phosphorylation and enhanced radioresistance. Thus, our data suggest that targeting signaling molecules such as PINCH1 that function downstream of focal adhesions (the complexes that mediate tumor cell adhesion to ECM) may overcome radio- and chemoresistance, providing new therapeutic approaches for cancer.
View details for DOI 10.1172/JCI41078
View details for PubMedID 20530873
View details for PubMedCentralID PMC2898588
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