The autophagy–lysosome pathway and apoptosis constitute vital determinants of cell fate and engage in a complex interplay in both physiological and pathological conditions. Central to this interplay is the archetypal autophagic cargo adaptor p62/SQSTM1/Sequestosome-1 which mediates both cell survival and endoplasmic reticulum stress-induced apoptosis via aggregation of ubiquitinated caspase-8. Here, we investigated the role of p62-mediated apoptosis in head and neck squamous cell carcinoma (HNSCC), which can be divided into two groups based on human papillomavirus (HPV) infection status. We show that increased autophagic flux and defective apoptosis are associated with radioresistance in HPV(-) HNSCC, whereas HPV(+) HNSCC fail to induce autophagic flux and readily undergo apoptotic cell death upon radiation treatments. The degree of radioresistance and tumor progression of HPV(-) HNSCC respectively correlated with autophagic activity and cytosolic levels of p62. Pharmacological activation of the p62-ZZ domain using small molecule ligands sensitized radioresistant HPV(-) HNSCC cells to ionizing radiation by facilitating p62 self-polymerization and sequestration of cargoes leading to apoptosis. The self-polymerizing activity of p62 was identified as the essential mechanism by which ubiquitinated caspase-8 is sequestered into aggresome-like structures, without which irradiation fails to induce apoptosis in HNSCC. Our results suggest that harnessing p62-dependent sequestration of ubiquitinated caspase-8 provides a novel therapeutic avenue in patients with radioresistant tumors.

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Inflammatory bowel disease (IBD), which includes both Crohn disease and ulcerative colitis, is a relapsing inflammatory disease of the gastrointestinal tract. Long-term chronic inflammatory conditions elevate the patient's risk of colorectal cancer (CRC). Currently, diagnosis requires endoscopy with biopsy. This procedure is invasive and requires a bowel-preparatory regimen, adding to patient burden. Interleukin 12 (IL12) and interleukin 23 (IL23) play key roles in inflammation, especially in the pathogenesis of IBD, and are established therapeutic targets. We propose that imaging of IL12/23 and its p40 subunit in IBD via immuno-PET potentially provides a new noninvasive diagnostic approach. Methods: Our aim was to investigate the potential of immuno-PET to image inflammation in a chemically induced mouse model of colitis using dextran sodium sulfate by targeting IL12/23p40 with a 89 Zr-radiolabeled anti-IL12/23p40 antibody. Results: High uptake of the IL12/23p40 immuno-PET agent was exhibited by dextran sodium sulfate-administered mice, and this uptake correlated with increased IL12/23p40 present in the sera. Competitive binding studies confirmed the specificity of the radiotracer for IL12/23p40 in the gastrointestinal tract. Conclusion: These promising results demonstrate the utility of this radiotracer as an imaging biomarker of IBD. Moreover, IL12/23p40 immuno-PET can potentially guide treatment decisions for IBD management.

Purpose: TRA-1-60 (TRA) is an established transcription factor of embryonic signaling and a well-known marker of pluripotency. It has been implicated in tumorigenesis and metastases, is not expressed in differentiated cells, which makes it an appealing biomarker for immunopositron emission tomography (immunoPET) imaging and radiopharmaceutical therapy (RPT). Herein, we explored the clinical implications of TRA in prostate cancer (PCa), examined the potential of TRA-targeted PET to specifically image TRA + cancer stem cells (CSCs) and assessed response to the selective ablation of PCa CSCs using TRA-targeted RPT. Experimental Design: First, we assessed the relationship between TRA (PODXL) copy number alterations (CNA) and survival using publicly available patient databases. The anti-TRA antibody, Bstrongomab, was radiolabeled with Zr-89 or Lu-177 for immunoPET imaging and RPT in PCa xenografts. Radiosensitive tissues were collected to assess radiotoxicity while excised tumors were examined for pathologic treatment response. Results: Patients with tumors having high PODXL CNA exhibited poorer progression-free survival than those with low PODXL, suggesting that it plays an important role in tumor aggressiveness. TRA-targeted immunoPET imaging specifically imaged CSCs in DU-145 xenografts. Tumors treated with TRA RPT exhibited delayed growth and decreased proliferative activity, marked by Ki-67 immunohistochemistry. Aside from minor weight loss in select animals, no significant signs of radiotoxicity were observed in the kidneys or livers. Conclusions: We successfully demonstrated the clinical significance of TRA expression in human PCa, engineered and tested radiotheranostic agents to image and treat TRA + prostate CSCs. Ablation of TRA + CSCs blunted PCa growth. Future studies combining CSC ablation with standard treatment will be explored to achieve durable responses.

DDR1 and DDR2 constitute a unique set of receptor tyrosine kinases that signal in response to collagen. Evidence indicates that DDRs play key roles in cancer progression. However, a comparison of the effects of DDR1 and DDR2 on tumor growth and metastasis within the same cellular context has not been done. To accomplish this goal, we developed a doxycycline (DOX) regulated system (Tet-Off) to express human DDR1b or DDR2 in the human HT1080 fibrosarcoma cell line. The cells were then examined for their ability to form subcutaneous (s.c.) tumors, or experimental lung metastases after intravenous (i.v.) injection in SCID mice. To induce or repress DDR expression in vivo, mice were fed a regular diet or a diet with DOX, respectively. Because collagen I (COL I) is a ligand for both DDR1 and DDR2, a set of mice were inoculated s.c. with HT1080 cells suspended in COL I with or without DDR induction in –DOX and +DOX mice, respectively. Growth of s.c. tumors was evaluated by measuring tumor volumes and lung metastases were quantitated by Alu qPCR and histological examination. In the absence of COL I, expression of either DDR1b or DDR2 had no impact on s.c. tumor growth rate, when compared to cells without DDR induction (+DOX). However, in the presence of COL I, tumor growth rates were significantly higher only in HT1080 cells with induced expression of DDR1b or DDR2 (p<0.001 for both), when compared to control cells inoculated with COL I into +DOX mice (no DDR induction). This suggests that a pro-oncogenic effect of DDR1b and DDR2 can be readily uncovered only in tumors growing within a COL I-rich environment. Because the Hippo tumor suppressor pathway has been shown to be regulated by extracellular matrix, homogenates of COL I tumors generated by cells with or without DDR induction were examined for levels and activation of Hippo pathway components. These analyses were consistent with Hippo pathway inactivation in tumors generated by DDR1b- and DDR2-expressing cells in a COL I matrix, consistent with their higher rate of tumor growth. Experimental metastasis assays revealed that induction of DDR2 expression had no impact on lung metastatic burden. In contrast, induction of DDR1b expression dramatically suppressed HT1080 lung metastasis formation, compared to control cells (no DDR1b induction). Collectively, these results suggest that: 1. Ectopic expression of DDRs in HT1080 cells accelerates tumor growth only when co-inoculated with COL I, consistent with a pro-tumorigenic effect of activated receptors within a collagen matrix. 2. A DDR/COL I axis supports HT1080 tumor growth, in part, by inactivation of the Hippo tumor suppressor pathway, suggesting that COL I-initiated DDR signaling regulates the Hippo pathway in vivo. 3. DDR1b, but not DDR2, is a potent suppressor of lung metastasis in the HT1080 model. Taken together, these studies provide novel insights into the complex roles of DDRs in cancer. Citation Format: Benjamin D. Wasinski, R. Daniel Bonfil, Anjum Sohail, Seong Ho Kim, Lisa Polin, Allen-Dexter Saliganan, Mohamad Bouhamdan, Sayed Nabi, Hyeong-Reh C. Kim, Marco Prunotto, Rafael A. Fridman. Complex roles of discoidin domain receptors (DDRs) in tumor growth and experimental metastasis: role of collagen I in DDR-mediated tumor growth [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 2135.

Background and aimsInflammatory bowel disease (IBD), which includes both Crohn’s Disease (CD) and ulcerative colitis (UC), is a relapsing inflammatory disease of the gastrointestinal (GI) tract. Long term chronic inflammatory conditions elevate patients’ risk for colorectal cancer (CRC). Currently, diagnosis requires endoscopy with biopsy. This procedure is invasive and requires bowel preparatory regimen, adding to patient burden. Interleukin 23 (IL23) plays a key role in inflammation especially in the pathogenesis of IBD and is an established therapeutic target. We propose that imaging of IL23 via immunopositron emission tomography (immunoPET) will potentially lead to a new non-invasive diagnostic approach.MethodsThe aim of the present study is to investigate the potential of immunoPET to image inflammation in a chemically induced mouse model of colitis using dextran sodium sulfate (DSS) by targeting IL23 via its p40 subunit with a89Zr-radiolabeled α-IL23p40 antibody.ResultsHigh uptake of the IL23p40 immunoPET agent in mice were displayed in DSS-administered mice, which correlated with increased IL23p40 present in sera. Competitive binding studies confirmed the specificity of the radiotracer for IL23p40 in the GI tract.ConclusionTaken together, these promising results set the stage for developing this radiotracer as an imaging biomarker for IBD diagnosis. Noninvasive imaging of IBD with IL23p40 immunoPET may help physicians in their treatment decisions for IBD management.