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Supplementary Data from β2-Microglobulin Signaling Blockade Inhibited Androgen Receptor Axis and Caused Apoptosis in Human Prostate Cancer Cells

Purpose: β2-Microglobulin (β2M) has been shown to promote osteomimicry and the proliferation of human prostate cancer cells. The objective of this study is to determine the mechanism by which targeting β2M using anti-β2M antibody inhibited growth and induced apoptosis in prostate cancer cells.Experimental Design: Polyclonal and monoclonal β2M antibodies were used to interrupt β2M signaling in human prostate cancer cell lines and the growth of prostate tumors in mice. The effects of the β2M antibody on a survival factor, androgen receptor (AR), and its target gene, prostate-specific antigen (PSA) expression, were investigated in cultured cells and in tumor xenografts.Results: The β2M antibody inhibited growth and promoted apoptosis in both AR-positive and PSA-positive, and AR-negative and PSA-negative, prostate cancer cells via the down-regulation of the AR in AR-positive prostate cancer cells and directly caused apoptosis in AR-negative prostate cancer cells in vitro and in tumor xenografts. The β2M antibody had no effect on AR expression or the growth of normal prostate cells.Conclusions: β2M downstream signaling regulates AR and PSA expression directly in AR-positive prostate cancer cells. In both AR-positive and AR-negative prostate cancer cells, interrupting β2M signaling with the β2M antibody inhibited cancer cell growth and induced its apoptosis. The β2M antibody is a novel and promising therapeutic agent for the treatment of human prostate cancers.

Objective: One of the unmet clinical challenges is that many tumors remain insensitive to radiotherapy owing to intrinsic resistance or acquired resistance that leads to tumor progression or recurrence. Moreover, radiation-induced acute injury and chronic inflammation in normal tissues limit the radiation dose that can be applied to the tumor, and tolerable doses are often linked to suboptimal tumor control—even accepting side effects that lead to decreased quality of life. Our goal of the study is to develop a nanoformulated radiosensitizer that inhibits NOX-ROS signal pathway to enhance therapeutic response in tumor cells while reducing radiotoxicity in normal tissues. Methods: We developed a biomaterial-based nanoparticle radiosensitizer that acts upon abnormal redox status and altered metabolism in tumor cells to trigger a cascade of changes in signal pathways to enhance response to radiotherapy. This CD44-targeted radiosensitizer consists of a biodegradable and bioactive hyaluronic acid nanoparticle (HANP) encapsulated with a dual NOX1/4 inhibitor, GKT831 (HANP/GKT831). Orthotopic and subcutaneous NSCLC tumor mouse models using a WRJ388 cell line that was isolated from a metastatic lung adenocarcinoma in a lymph node of a KrasG12D/Lkb1null GEMM mouse were established for evaluation of the effect on the inhibition of tumor growth and protection of normal tissue after the treatment with intravenous administrations of HANP/GKT831 at 5 mg/kg (twice per week for five times) combined with radiation (2 Gy/each dose, twice per week for five times). The anti-tumor effect, immune response, and inflammation in the normal lung tissues were investigated using histological and immunofluorescence analyses. Results: Our results showed that HANP/GKT831 had stronger inhibitory effects on ROS generation and cell proliferation than that of GKT831 in mouse NSCLC tumor cells in vitro. Systemic injection of HANP/GKT831 combined with radiation had the strongest inhibition on tumor growth (~79%) compared with the combination of non-nanoformulated GKT831 with radiation treated mouse group ( Conclusion: HANP/GKT831 is a biomaterial-based nanoparticle radiosensitizer that acts upon abnormal redox status and altered metabolism in tumor cells to trigger a cascade of changes in signal pathways to enhance the response of tumor cells to radiotherapy. It also has the potential to reduce the radiation-induced inflammatory response in normal lung tissues. Citation Format: Lei Zhu, Lumeng Zhang, Tongrui Liu, Wei Ping Qian, Wei Zhou, Lily Yang. Development of a NOX-ROS nanoinhibitor for enhancing response to radiotherapy in lung cancer cells while mitigation radiation-induced inflammation and injury in the normal lung tissue [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 811.

Objectives: Resistance to therapy is the major unmet challenge in clinical management of breast cancer. NADPH oxidases 4 (NOX4) is a critical NADPH oxidase subunit because it constitutively reacts with hydrogen peroxide (H2O2), generating ROS that is associated with tumorigenesis, metastasis, and invasion. A high level of NOX4 activity has been associated with drug resistance in human cancers. To improve the therapeutic efficacy, we have developed a hyaluronic acid nanoparticle (HANP) carrying a NOX4 inhibitor and examined the anti-tumor effect in combination with radiotherapy (RT) or chemotherapy. Methods: Biodegradable polymeric HANP was synthesized and encapsulated with GKT831 (HANP/GKT831), a novel inhibitor of NOX4. Targeted delivery of GKT831 with HANP was first studied by optical imaging in an orthotopic human breast cancer patient tissue derived xenograft (PDX) model. Subsequently, the ability of HANP/GKT831 in inhibition of tumor cells and sensitization of tumor cells to chemotherapy and RT was evaluated in breast PDX models following intravenous administration of HANP/GKT831 (containing 5 mg/kg equivalent dose of GKT831) in combination with Doxorubicin (DOX, 5 mg/kg) and RT (2 Gy) for 5 times, once per week. Furthermore, mechanism of NOX4 inhibition on improving therapeutic response in breast cancer was investigated. Results: We have developed HANP/GKT831 with drug loading efficiency of 15% (w/w) determined by high performance liquid chromatography (HPLC). The selective accumulation of HANP/GKT831 in breast PDX tumors following systemic delivery was demonstrated by optical imaging. Following five treatments, nude mice bearing breast PDX tumors that received HANP/GKT831 in combination with DOX or RT had significant stronger tumor growth inhibition compared with either DOX or RT alone (73.71±13.87% and 79.33±19%, respectively) as well as the non-treatment control group (86.33±16.25% and 78.15±14.27%, respectively). Examination of tumor tissues revealed that the level of poly (ADP-ribose) polymerase (PARP) expression was reduced in tumors treated with HANP/GKT831 in combination of RT or DOX, which induces cell death through DNA damage. Conclusions: We have developed a polymeric HANP carrying a NOX4 inhibitor (HANP/GKT831). We found that HANP/GKT831 could be efficiently delivered into tumors following systemic administration and significantly enhanced tumor response to Dox treatment or radiotherapy in two multi-drug resistant breast PDX tumor models. Therefore, HANP/GKT831 is a promising therapy agent for targeted therapy of breast cancer. Currently, mechanism of the effect on chemo- and radiotherapy sensitization is under investigation. One of the possible mechanisms is the downregulation of DNA-damage repairing functions, such as downregulation of DNA damage repair enzyme PARP. Citation Format: Lei Zhu, Yi Zhao, Wei Ping Qian, Dazhi Wang, Bing-Hua Jiang, Lily Yang. NADPH oxidases 4 inhibition using a hyaluronic acid nanoparticle drug delivery system sensitized therapeutic response to chemo and radiotherapy in drug resistant breast cancer [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 3933.