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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.

Upregulation of NADPH oxidases (NOXs) in cancer cells leads to chronic increase in intracellular reactive oxygen species (ROS) and adaptation to a high ROS level for cell survival and, thereby, low sensitivity to radiotherapy. To overcome resistance to radiotherapy, we have developed a bioactive and CD44 targeted hyaluronic acid nanoparticle encapsulated with an NOX inhibitor, GKT831 (HANP/GKT831). We found that HANP/GKT831 had stronger inhibitory effects on ROS generation and cell proliferation than that of GKT831 alone in cancer cells. Systemic delivery of HANP/GKT831 led to the targeted accumulation in breast cancer patient derived xenograft (PDX) tumors in nude mice. Importantly, the combination of systemic delivery of HANP/GKT831 with a low dose of local radiotherapy significantly enhanced tumor growth inhibition in breast cancer PDX models. Our results showed that HANP/GKT831 primed tumor cells to radiation-induced DNA damage and cell death by downregulation of DNA repair function and oncogenic signal pathways.

RNA splicing plays significant roles in fundamental biological activities. However, our knowledge about the roles of alternative splicing and underlying mechanisms during spermatogenesis is limited. Here, we report that Serine/arginine-rich splicing factor 2 (SRSF2), also known as SC35, plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf2 by Stra8-Cre caused complete infertility and defective spermatogenesis. Further analyses revealed that deletion of Srsf2 disrupted differentiation and meiosis initiation of spermatogonia. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF2 regulatory networks play critical roles in several major events including reproductive development, spermatogenesis, meiotic cell cycle, synapse organization, DNA recombination, chromosome segregation, and male sex differentiation. Furthermore, SRSF2 affected expression and alternative splicing of Stra8, Stag3 and Atr encoding critical factors for spermatogenesis in a direct manner. Taken together, our results demonstrate that SRSF2 has important functions in spermatogenesis and male fertility by regulating alternative splicing.

Mammalian oocytes are arrested at the diplotene stage of prophase I during fetal or postnatal development. It was reported that cyclin-dependent kinases (CDK1) was the sole CDK to drive the resumption of meiosis and CDK2 was dispensable for meiosis progression in mouse oocytes according to the conditional knockout studies. However, a recent study showed that CDK2 activity is essential for meiotic division and gametogenesis by means of gene-directed mutagenesis, which avoids the compensatory activation of other CDKs. Taken the compensatory effect between CDKs after gene knockout, the physiological function of CDK2 activity in oocyte maturation remains unclear. To address this issue, we applied a specific small-molecule inhibitor to restrain CDK2 activity transiently during oocyte meiotic maturation. Surprisingly, transient inhibition of CDK2 activity severely prevented the meiosis I completion although the meiotic resumption was not affected. Then we found that CDK2 activity was required for establishment of normal spindle and chromosome dynamics. Notably, CDK2 inhibition interrupted the anaphase-promoting complex/cyclosome (APC/C)-dependent degradation pathway by maintaining the activation of spindle assembly checkpoint (SAC). Interestingly, CDK2 inhibition prevented the egg activation as well. Overall, our data demonstrate that CDK2 kinase activity is required for proper dynamics of spindle and chromosomes, whose disturbance induces the continuous SAC activation and subsequent inactivation of APC/C activity in oocyte meiosis.

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.

Patulin is a secondary metabolite mainly secreted by fungi and is the most common mycotoxin found in apples and apple-based products. For the past few years, numerous studies suggested the wide distribution and toxicity of patulin. In this study, we investigated the toxic effect of patulin on mouse oocytes and its possible mechanisms. The results showed that patulin treatment did not affect meiotic resumption, but inhibited oocyte maturation as indicated by failure of first polar body extrusion. Further mechanistic study showed that patulin treatment disturbed normal spindle assembly, chromosome alignment and morphology. We also found increased oxidative stress by testing the level of ROS and decreased mitochondrial membrane potential, indicating mitochondria dysfunction. In summary, our results suggest that patulin treatment causes oocyte meiotic arrest by disturbing normal spindle assembly and chromosome alignment, which may be caused by dysfunctions of mitochondria.