Machine learning-based prediction model and visual interpretation for prostate cancer.

BACKGROUND: Most prostate cancers(PCa) rely on serum prostate-specific antigen (PSA) testing for biopsy confirmation, but the accuracy needs to be further improved. We need to continue to develop PCa prediction model with high clinical application value. METHODS: Benign prostatic hyperplasia (BPH) and prostate cancer data were obtained from the Chinese National Clinical Medical Science Data Center for retrospective analysis. The model was constructed using the XGBoost algorithm, and patients' age, body mass index (BMI), PSA-related parameters and serum biochemical parameters were used as model variables. Using decision analysis curve (DCA) to evaluate the clinical utility of the models. The shapley additive explanation (SHAP) framework was used to analyze the importance ranking and risk threshold of the variables. RESULTS: A total of 1915 patients were included in this study, including 823 (43.0%) were BPH patients and 1092 (57.0%) were PCa patients. The XGBoost model provided better performance (AUC 0.82) compared with f/tPSA (AUC 0.75),tPSA (AUC 0.68) and fPSA (AUC 0.61), respectively. Based on SHAP values, f/tPSA was the most important variable, and the top five most important biochemical parameter variables were inorganic phosphorus (P), potassium (K), creatine kinase MB isoenzyme (CKMB), low-density lipoprotein cholesterol (LDL-C), and creatinine (Cre). PCa risk thresholds for these risk markers were f/tPSA (0.13), P (1.29 mmol/L), K (4.29 mmol/L), CKMB ( 11.6U/L), LDL-C (3.05mmol/L) and Cre (74.5-99.1umol/L). CONCLUSION: The present model has advantages of wide-spread availability and high net benefit, especially for underdeveloped countries and regions. Furthermore, these risk thresholds can assist in the diagnosis and screening of prostate cancer in clinical practice.

SRSF3 and HNRNPH1 Regulate Radiation-Induced Alternative Splicing of Protein Arginine Methyltransferase 5 in Hepatocellular Carcinoma.

Protein arginine methyltransferase 5 (PRMT5) is an epigenetic regulator which has been proven to be a potential target for cancer therapy. We observed that PRMT5 underwent alternative splicing (AS) and generated a spliced isoform PRMT5-ISO5 in hepatocellular carcinoma (HCC) patients after radiotherapy. However, the regulatory mechanism and the clinical implications of IR-induced PRMT5 AS are unclear. This work revealed that serine and arginine rich splicing factor 3 (SRSF3) silencing increased PRMT5-ISO5 level, whereas heterogeneous nuclear ribonucleoprotein H 1 (HNRNPH1) silencing reduced it. Then, we found that SRSF3 and HNRNPH1 competitively combined with PRMT5 pre-mRNA located at the region around the 3'- splicing site on intron 2 and the alternative 3'- splicing site on exon 4. IR-induced SRSF3 downregulation led to an elevated level of PRMT5-ISO5, and exogenous expression of PRMT5-ISO5 enhanced cell radiosensitivity. Finally, we confirmed in vivo that IR induced the increased level of PRMT5-ISO5 which in turn enhanced tumor killing and regression, and liver-specific Prmt5 depletion reduced hepatic steatosis and delayed tumor progression of spontaneous HCC. In conclusion, our data uncover the competitive antagonistic interaction of SRSF3 and HNRNPH1 in regulating PRMT5 splicing induced by IR, providing potentially effective radiotherapy by modulating PRMT5 splicing against HCC.

AdipoR1 Regulates Ionizing Radiation-Induced Ferroptosis in HCC cells through Nrf2/xCT Pathway.

Radiotherapy has been used for decades in the treatment of liver cancer. We previously found that adiponectin receptor (AdipoR1) is a prognostic biomarker for hepatoma carcinoma (HCC) after stereotactic body radiation therapy (SBRT) and blocking AdipoR1 enhances radiation sensitivity in hepatoma carcinoma cells. In the current study, we aimed to elucidate the roles of AdipoR1 in ionizing radiation- (IR-) induced radiosensitivity by activating ferroptosis pathway in HCC cells. We found that IR upregulated the expression of AdipoR1 and furthermore promoted the protein stability of transcription factor Nrf2, Nrf2 binded to the xCT promoter and increased xCT transcription and expression, and this directly contributed to the protective function in the early stage of radiation in HCC cells. AdipoR1 knockdown significantly inhibited expression of Nrf2 and xCT and, furthermore, increased both IR- and erastin-induced ferroptosis, which could be abolished by the rescue of Nrf2 and xCT. For the first time, we found that radiation-induced ferroptosis was mediated by AdipoR1-Nrf2-xCT pathway in HCC cells. These results provide new insights to the development and application of novel therapeutic strategies for hepatoma carcinoma.

ESR1 inhibits ionizing radiation-induced ferroptosis in breast cancer cells via the NEDD4L/CD71 pathway.

Ferroptosis is the name given to the type of non-apoptotic cell death that is caused by iron accumulation and subsequent lipid peroxidation. However, how ionizing radiation (IR)-induced ferroptosis is regulated in estrogen receptor-positive (ER+) breast cancer cells remains unclear. To attempt to resolve this issue, bioinformatics analysis was performed to evaluate the prognostic value of estrogen receptor 1 (ESR1) in breast cancer tissues. A total of four breast cancer cell lines and an MCF10A non-malignant counterpart were used. Western blotting was used to analyze the levels of protein expression, whereas immunoprecipitation (IP) and ubiquitination experiments were used to test protein binding and ubiquitination levels, respectively. Flow cytometry was subsequently used to analyze cell death and lipid peroxidation levels. The results showed that a high expression level of ESR1 was significantly correlated with poor overall survival in breast cancer. ESR1 knockdown significantly enhanced IR-induced ferroptosis and increased the CD71 protein level. The IP results showed that ESR1 enhanced the binding of the E3 ubiquitin ligase NEDD4L to CD71, promoting the ubiquitination and degradation of CD71, suggesting that CD71 expression was regulated by both ESR1 and NEDD4L. Taken together, the findings in the present study have demonstrated a regulatory relationship between ESR1 and NEDD4L/CD71 in IR-induced ferroptosis. In addition, the ESR1/NEDD4L/CD71 axis may be a potential target for the radiotherapy of breast cancer.

Iron-Dependent Autophagic Cell Death Induced by Radiation in MDA-MB-231 Breast Cancer Cells.

In radiation oncology, ionizing radiation is used to kill cancer cells, in other words, the induction of different types of cell death. To investigate this cellular death and the associated iron accumulation, the transfer, release, and participation of iron after radiation treatment was analyzed. We found that radiation-induced cell death varied in different breast cancer cells and autophagy was induced in MDA-MB-231 and BT549 cells (triple negative breast cancer cell line) rather than in MCF-7 and zr-75 cells. Iron chelator deferoxamine (DFO), the autophagy inhibitor 3MA, silencing of the autophagy-related genes ATG5, and Beclin 1 could decrease radiation induced cell death in MDA-MB-231 cells, while inhibitors of apoptosis such as Z-VAD-FMK, ferroptosis inhibitor ferrostatin-1 (Fer-1), and necroptosis inhibitor Necrostatin-1 showed no change. This suggests the occurrence of autophagic cell death. Furthermore, we found that iron accumulation and iron regulatory proteins, including transferrin (Tf), transferrin receptor (CD71), and Ferritin (FTH), increased after radiation treatment, and the silencing of transferrin decreased radiation-induced cell death. In addition, radiation increased lysosomal membrane permeabilization (LMP) and the release of lysosomal iron and cathepsins, while cathepsins silencing failed to change cell viability. Radiation-induced iron accumulation increased Reactive oxygen species (ROS) generation via the Fenton reaction and increased autophagy in a time-dependent manner. DFO, N-acetylcysteine (NAC), and overexpression of superoxide dismutase 2 (SOD2) decreased ROS generation, autophagy, and cell death. To summarize, for the first time, we found that radiation-induced autophagic cell death was iron-dependent in breast cancer MDA-MB-231 cells. These results provide new insights into the cell death process of cancers and might conduce to the development and application of novel therapeutic strategies for patients with apoptosis-resistant breast cancer.

p53-Induced Autophagy Regulates Chemotherapy and Radiotherapy Resistance in Multidrug Resistance Cancer Cells.

BACKGROUND: Multidrug resistance (MDR), a major problem in oncology therapy, limits the effectiveness of anticancer drugs. Although p53 functions as a tumor suppressor, the associations between p53 status, autophagy, and MDR are complicated and conditional. METHOD:  In this report, p53-null human ovarian cancer cell line SKOV3 and its MDR phenotype SKVCR and human leukemia cell line CEM and its MDR phenotype CEM-VLB) (p53 mutant cell line) were used. RESULTS:  Compared to parental SKOV3, the mRNA and protein levels of MAPLC3-II and Beclin1 were higher in SKVCR cells. The inhibition of autophagy by 3-MA significantly sensitized SKVCR to VCR. Conversely, in drug-resistant leukemic cells CEM-VLB, the expressions of Beclin1 and MAPLC3-II were lower than CEM. CEM and CEM-VLB cells were treated with VLB .01 or 0.5 µg/mL, respectively, and the expression of p53 and autophagy up-regulated after VLB (.01 µg/mL) treatment in CEM cells. The percentage of S-phase and G2/M phase cells up-regulated significantly by .01 µg/mL VLB in CEM, which may relate to the status of p53 of CEM cells. A combination of radiation with 3-MA significantly increased apoptosis in CEM-VLB cells. CONCLUSION:  Our discovery found that p53 is an important regulator controlling the balance between autophagy and MDR, as a potential drug target for ovarian cancer and leukemia.

Valproic Acid Regulates HR and Cell Cycle Through MUS81-pRPA2 Pathway in Response to Hydroxyurea.

Breast cancer is the primary problem threatening women's health. The combined application of valproic acid (VPA) and hydroxyurea (HU) has a synergistic effect on killing breast cancer cells, but the molecular mechanism remains elusive. Replication protein A2 phosphorylation (pRPA2), is essential for homologous recombination (HR) repair and cell cycle. Here we showed that in response to HU, the VPA significantly decreased the tumor cells survival, and promoted S-phase slippage, which was associated with the decrease of pCHK1 and WEE1/pCDK1-mediated checkpoint kinases phosphorylation pathway and inhibited pRPA2/Rad51-mediated HR repair pathway; the mutation of pRPA2 significantly diminished the above effect, indicating that VPA-caused HU sensitization was pRPA2 dependent. It was further found that VPA and HU combination treatment also resulted in the decrease of endonuclease MUS81. After MUS81 elimination, not only the level of pRPA2 was abolished in response to HU treatment, but also VPA-caused HU sensitization was significantly down-regulated through pRPA2-mediated checkpoint kinases phosphorylation and HR repair pathways. In addition, the VPA altered the tumor microenvironment and reduced tumor burden by recruiting macrophages to tumor sites; the Kaplan-Meier analysis showed that patients with high pRPA2 expression had significantly worse survival. Overall, our findings demonstrated that VPA influences HR repair and cell cycle through down-regulating MUS81-pRPA2 pathway in response to HU treatment.

The Valproate Mediates Radio-Bidirectional Regulation Through RFWD3-Dependent Ubiquitination on Rad51.

Ionizing radiation (IR) can induce DNA double-strand breaks (DSBs) in tumor cells during radiotherapy (RT), but the efficiency of RT is limited because of the toxicity to normal cells. Locating an adjuvant treatment to alleviate damage in normal cells while sensitizing tumor cells to IR has attracted much attention. Here, using the 7,12-dimethylbenz[α]anthracene (DMBA)-induced malignant transformed MCF10A cells, we found that valproate (VPA), a histone deacetylase inhibitor (HDACi), radiosensitized transformed cells while alleviated IR-induced damage in normal cells at a safe dose (0.5 mM). We further demonstrated the decrease of homologous recombination (HR)-associated Rad51 in the transformed cells was related to the increase of its ubiquitination regulated by E3 ligase RFWD3 for the radiosensitization, which was opposite to normal cells, indicating that RFWD3-dependent ubiquitination on Rad51 was involved in the VPA-mediated radio-bidirectional effect. Through DMBA-transformed breast cancer rat model, VPA at 200 mg/kg radiosensitized tumor tissue cells by increasing RFWD3 and inhibited Rad51, while radioprotected normal tissue cells by decreasing RFWD3 and enhanced Rad51. In addition, we found high-level Rad51 was associated with tumorigenesis and poor prognosis in breast cancer patients. Our findings uncovered RFWD3-dependent Rad51 ubiquitination was the novel mechanism of VPA-mediated radio-bidirectional effect, VPA is a potential adjuvant treatment for tumor RT.

Furazolidone Increases Survival of Mice Exposed to Lethal Total Body Irradiation through the Antiapoptosis and Antiautophagy Mechanism.

Exposure to total body irradiation (TBI) causes dose- and tissue-specific lethality. However, there are few effective and nontoxic radiation countermeasures for the radiation injury. In the current study, mice were pretreated with a traditional antimicrobial agent, FZD, before TBI; the protective effects of FZD on radiation injury were evaluated by using parameters such as the spleen index and thymus index, immunohistochemical staining of intestinal tissue, and frequency of micronuclei in polychromatophilic erythrocytes of bone marrow. The intestinal epithelial cell line IEC-6 was used to investigate the underlying mechanisms. Our results indicated that FZD administration significantly improved the survival of lethal dose-irradiated mice, decreased the number of micronuclei, upregulated the number of leukocytes and immune organ indices, and restored intestinal integrity in mice after TBI. TUNEL and western blot showed that FZD protected intestinal tissue by downregulating radiation-induced apoptosis and autophagy. Meanwhile, FZD protected IEC-6 cells from radiation-induced cell death by inhibiting apoptosis and autophagy. To sum up, FZD protected against radiation-induced cell death both in vitro and in vivo through antiapoptosis and antiautophagy mechanisms.

Valproic acid sensitizes breast cancer cells to hydroxyurea through inhibiting RPA2 hyperphosphorylation-mediated DNA repair pathway.

It was reported that valproic acid (VPA, a histone deacetylase inhibitor) can sensitize cancer cells to hydroxyurea (HU, a ribonucleotide reductase inhibitor) for chemotherapy, although the mechanism of VPA-induced HU sensitization is unclear. In this study, we systematically characterized VPA-induced HU sensitization of breast cancer cells. Multiple breast cancer cell models were employed to investigate whether the safe concentration of 0.5mM VPA and 2mM HU can result in DNA double-strand breaks (DSBs) and impact cell survival. Furthermore, the underlying mechanism was explored through cell biology assays, including clonogenic survival, homologous recombination (HR) activity, immunoblot and immunofluorescence. We found that VPA and HU cooperatively suppressed cancer cell survival. VPA resulted in the accumulation of more DNA double-strand breaks (DSBs) in response to HU-induced replication arrest and was able to block HU-stimulated homologous recombination (HR) through inhibiting the activity of two key HR repair proteins by hyperphosphorylation of replication protein A2 (RPA2-p) and recombinase Rad51. However, apoptosis was not detected under this condition. In addition, the results from the survival fraction in the cells expressing defective RPA2-p showed that VPA disrupted the HU-induced RPA2-p-Rad51-mediated HR pathway. Importantly, these findings were further supported by analyzing primary-culture cells from the tissue of chemical carcinogen (DMBA)-induced breast cancer in rats. Thus, our data demonstrated that VPA and HU synergistically suppressed tumor cells via disturbing RPA2-p-mediated DNA repair pathway, which provides a new way for combining chemotherapeutic drugs to sensitize breast cancer cells.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats.

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function.