Purpose: Triple-negative breast cancers (TNBC) are defined by a lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (ERBB2/HER2). Although initially responsive to chemotherapy, most recurrent TNBCs develop resistance, resulting in disease progression. Autophagy is a lysosome-mediated degradation and recycling process that can function as an adaptive survival response during chemotherapy and contribute to chemoresistance. Our goal was to determine whether autophagy inhibition improves treatment efficacy in TNBC cells in tumors either sensitive or refractory to anthracyclines. Experimental Design: We used in vitro and in vivo models of TNBC using cell lines sensitive to epirubicin and other anthracyclines, as well as derivative lines, resistant to the same drugs. We assessed basal autophagy levels and the effects of chemotherapy on autophagy in parental and resistant cells. Applying various approaches to inhibit autophagy alone and in combination with chemotherapy, we assessed the effects on cell viability in vitro and tumor growth rates in vivo. Results: We demonstrated that epirubicin induced autophagic flux in TNBC cells. Epirubicin-resistant lines exhibited at least 1.5-fold increased basal autophagy levels and, when treated with autophagy inhibitors, showed a significant loss in viability, indicating dependence of resistant cells on autophagy for survival. Combination of epirubicin with the autophagy inhibitor hydroxychloroquine resulted in a significant reduction in tumor growth compared with monotherapy with epirubicin. Conclusion: Autophagy inhibition enhances therapeutic response in both anthracycline-sensitive and -resistant TNBC and may be an effective new treatment strategy for this disease. Clin Cancer Res; 20(12); 3159–73. ©2014 AACR.

Background: Methods: Results: Conclusion:

Integrin-linked kinase (ILK) was assesed as a therapeutic target in glioblastoma xenograft models through multiple endpoints including treatment related changes in the tumor microenvironment. Glioblastoma cell lines were tested in vitro for sensitivity toward the small-molecule inhibitors QLT0254 and QLT0267. Cell viability, cell cycle, and apoptosis were evaluated using MTT assay, flow cytometry, caspase activation, and DAPI staining. Western blotting and ELISA were used for protein analysis (ILK, PKB/Akt, VEGF, and HIF-1α). In vivo assessment of growth rate, cell proliferation, BrdUrd, blood vessel mass (CD31 labeling), vessel perfusion (Hoechst 33342), and hypoxia (EF-5) was done using U87MG glioblastoma xenografts in RAG2-M mice treated orally with QLT0267 (200 mg/kg q.d.). ILK inhibition in vitro with QLT0254 and QLT0267 resulted in decreased levels of phospho-PKB/Akt (Ser⁴⁷³), secreted VEGF, G₂-M block, and apoptosis induction. Mice treated with QLT0267 exhibited significant delays in tumor growth (treated 213 mm³ versus control 549 mm³). In situ analysis of U87MG tumor cell proliferation from QLT0267-treated mice was significantly lower relative to untreated mice. Importantly, VEGF and HIF-1α expression decreased in QLT0267-treated tumors as did the percentage of blood vessel mass and numbers of Hoechst 33342 perfused tumor vessels compared with control tumors (35% versus 83%). ILK inhibition with novel small-molecule inhibitors leads to treatment-associated delays in tumor growth, decreased tumor angiogenesis, and functionality of tumor vasculature. The therapeutic effects of a selected ILK inhibitor (QLT0267) should be determined in the clinic in cancers that exhibit dysregulated ILK, such as PTEN-null glioblastomas. [Mol Cancer Ther 2008;7(1):59–70]

Glutathione is the predominant endogenous cellular antioxidant, playing a critical role in the cellular defensive response to oxidative stress by neutralizing free radicals and reactive oxygen species. With cysteine as the rate-limiting substrate in glutathione biosynthesis, the cystine/glutamate transporter (system x_c^-) represents a potentially attractive PET biomarker to enable in vivo quantification of x_c^- activity in response to oxidative stress associated with disease. We have developed a system x_c^- substrate that incorporates characteristics of both natural substrates, l-cystine and l-glutamate (l-Glu). l-aminosuberic acid (l-ASu) has been identified as a more efficient system x_c^- substrate than l-Glu, leading to an assessment of a series of anionic amino acids as prospective PET tracers. Herein, we report the synthesis and in vitro and in vivo validation of a lead candidate, ¹⁸F-5-fluoro-aminosuberic acid (¹⁸F-FASu), as a PET tracer for functional imaging of a cellular response to oxidative stress with remarkable tumor uptake and retention. Methods: ¹⁸F-FASu was identified as a potential PET tracer based on an in vitro screening of compounds similar to l-cystine and l-Glu. Affinity toward system x_c^- was determined via in vitro uptake and inhibition studies using oxidative stress–induced EL4 and SKOV-3 cells. In vivo biodistribution and PET imaging studies were performed in mice bearing xenograft tumors (EL4 and SKOV-3). Results: In vitro assay results determined that l-ASu inhibited system x_c^- as well as or better than l-Glu. The direct comparison of uptake of tritiated compounds demonstrated more efficient system x_c^- uptake of l-ASu than l-Glu. Radiosynthesis of ¹⁸F-FASu allowed the validation of uptake for the fluorine-bearing derivative in vitro. Evaluation in vivo demonstrated primarily renal clearance and uptake of approximately 8 percentage injected dose per gram in SKOV-3 tumors, with tumor-to-blood and tumor-to-muscle ratios of approximately 12 and approximately 28, respectively. ¹⁸F-FASu uptake was approximately 5 times greater than ¹⁸F-FDG uptake in SKOV-3 tumors. Dynamic PET imaging demonstrated uptake in EL4 tumor xenografts of approximately 6 percentage injected dose per gram and good tumor retention for at least 2 h after injection. Conclusion: ¹⁸F-FASu is a potentially useful metabolic tracer for PET imaging of a functional cellular response to oxidative stress. ¹⁸F-FASu may provide more sensitive detection than ¹⁸F-FDG in certain tumors.