TP53-specific mutations serve as a potential biomarker for homologous recombination deficiency in breast cancer: a clinical next-generation sequencing study.

Background: TP53 mutations and homologous recombination deficiency (HRD) occur frequently in breast cancer. However, the characteristics of TP53 pathogenic mutations in breast cancer patients with/without HRD are not clear. Methods: Clinical next-generation sequencing (NGS) of both tumor and paired blood DNA from 119 breast cancer patients (BRCA-119 cohort) was performed with a 520-gene panel. Mutations, tumor mutation burden (TMB), and genomic HRD scores were assessed from NGS data. NGS data from 47 breast cancer patients in the HRD test cohort were analyzed for further verification. Results: All TP53 pathogenic mutations in patients had somatic origin, which was associated with the protein expression of estrogen receptor and progestogen receptor. Compared to patients without TP53 pathologic mutations, patients with TP53 pathologic mutations had higher levels of HRD scores and different genomic alterations. The frequency of TP53 pathologic mutation was higher in the HRD-high group (HRD score ≥ 42) relative to that in the HRD-low group (HRD score < 42). TP53 has different mutational characteristics between the HRD-low and HRD-high groups. TP53-specific mutation subgroups had diverse genomic features and TMB. Notably, TP53 pathogenic mutations predicted the HRD status of breast cancer patients with an area under the curve (AUC) of 0.61. TP53-specific mutations, namely HRD-low mutation, HRD-high mutation, and HRD common mutation, predicted the HRD status of breast cancer patients with AUC values of 0.32, 0.72, and 0.58, respectively. Interestingly, TP53 HRD-high mutation and HRD common mutation combinations showed the highest AUC values (0.80) in predicting HRD status. Conclusions: TP53-specific mutation combinations predict the HRD status of patients, indicating that TP53 pathogenic mutations could serve as a potential biomarker for poly-ADP-ribose polymerase (PARP) inhibitors in breast cancer patients .

Somatic mutations in four novel genes contribute to homologous recombination deficiency in breast cancer: a real-world clinical tumor sequencing study.

Breast cancers involving mutations in homologous recombination (HR) genes, most commonly BRCA1 and BRCA2 (BRCA1/2), respond well to PARP inhibitors and platinum-based chemotherapy. However, except for these specific HR genes, it is not clear which other mutations contribute to homologous recombination defects (HRD). Here, we performed next-generation sequencing of tumor tissues and matched blood samples from 119 breast cancer patients using the OncoScreen Plus panel. Genomic mutation characteristics and HRD scores were analyzed. In the HR genes, we found that BRCA1/2 and PLAB2 mutations were related to HRD. HRD was also detected in a subset of patients without germline or somatic mutations in BRCA1/2, PLAB2, or other HR-related genes. Notably, LRP1B, NOTCH3, GATA2, and CARD11 (abbreviated as LNGC) mutations were associated with high HRD scores in breast cancer patients. Furthermore, functional experiments demonstrated that silencing CARD11 and GATA2 impairs HR repair efficiency and enhances the sensitivity of tumor cells to olaparib treatment. In summary, in the absence of mutations in the HR genes, the sensitivity of tumor cells to PARP inhibitors and platinum-based chemotherapy may be enhanced in a subset of breast cancer patients with LNGC somatic mutations.

Upregulated YTHDF1 associates with tumor immune microenvironment in head and neck squamous cell carcinomas.

Background: The nature of the tumor immune microenvironment (TME) is essential for the head and neck squamous cell carcinomas (HNSCC) initiation, prognosis, and response to immunotherapy. However, its gene regulatory network remains to be elucidated. Methods: To identify N6-methyladenosine (m6A) regulators that are involved in regulating the HNSCC TME, a computational screen was applied to The Cancer Genome Atlas (TCGA) HNSCC patient samples. The effects of mutation, copy number variation (CNV), and transcriptional regulation on YTHDF1 expression were analyzed. We analyzed the TME infiltration, cancer-immunity cycle activities, and YTHDF1-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Results: Among the 24 m6A regulators, 3 factors (YTHDF1, ELAVL1, and METTL3) were highly correlated with TME infiltration. As the top candidate, YTHDF1 was up-regulated and amplified in HNSCC. YTHDF1 promoter gains active histone marks and high chromatin accessibility, which might be transcriptionally activated by SOX2 and TP63. Moreover, YTHDF1 expression significantly associates with tumor malignant phenotype in HNSCC, which has a positive correlation with CD4+ T cells and a negative correlation with CD8+ T cells infiltration. Specifically, YTHDF1 expression is negatively associated with the cancer-immunity cycle and immune checkpoint inhibitors. In terms of the underlying biological mechanisms, YTHDF1 may interact with YTHDF2/3 to regulate several vital immune-related pathways. Conclusions: We identify YTHDF1 associated with TME and elucidate an underlying mechanism of immune escape in HNSCC, which might be used as a predictive marker in guiding immunotherapy.

Expression Pattern and Prognostic Value of CTLA-4, CD86, and Tumor-Infiltrating Lymphocytes in Rectal Cancer after Neoadjuvant Chemo(radio)therapy.

The synergistic effect of combining immune checkpoint inhibitors (ICIs) with neoadjuvant chemo(radio)therapy (nCRT) in colorectal cancer is still limited. We aimed to understand the impact of nCRT on the tumor microenvironment and to explore favorable immune markers of this combination. Herein, we investigated the expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), CD86, CD4, and CD8 after nCRT and its association with clinicopathological characteristics. Immunostaining of immune-related molecules was performed in 255 surgically resected specimens from rectal cancer patients treated with nCRT. CD4 and CD8 expression on the tumor (tCD4/CD8), stroma (sCD4/CD8), and invasive front (iCD4/CD8) was evaluated. The expression levels of immune-related molecules were significantly lower in the nCRT-treated group, except for CTLA-4 and sCD8. However, patients with higher sCD8+ cell density and CTLA-4 expression had better progression-free survival (PFS) and distant metastasis-free survival (DMFS). In addition, higher CD86 expression was associated with poorer overall survival (OS). Higher CTLA-4 expression was associated with higher tCD8+ cell density, whereas CD86 expression was correlated with the cell density of t/sCD8. Prognostic analysis confirmed that the relationships between CTLA-4 and DMFS as well as CD86 and OS were significantly correlated in low rather than high CD8+ cell density. Further the combination of CD8+ cell density and CD86 expression was shown to be an independent prognostic factor of OS, whereas the combination of CTLA-4 was not for DMFS. Together, these results demonstrate significant correlations between CD86 expression and t/sCD8+ cell density in rectal cancer after nCRT and could potentially have clinical implications for combining ICIs and nCRT.

NOP53 undergoes liquid-liquid phase separation and promotes tumor radio-resistance.

Aberrant DNA damage response (DDR) axis remains the major molecular mechanism for tumor radio-resistance. We recently characterized liquid-liquid phase separation (LLPS) as an essential mechanism of DDR, and identified several key DDR factors as potential LLPS proteins, including nucleolar protein NOP53. In this study, we found that NOP53 formed highly concentrated droplets in vivo and in vitro, which had liquid-like properties including the fusion of adjacent condensates, rapid fluorescence recovery after photobleaching and the sensitivity to 1,6-hexanediol. Moreover, the intrinsically disordered region 1 (IDR1) is required for NOP53 phase separation. In addition, multivalent-arginine-rich linear motifs (M-R motifs), which are enriched in NOP53, were essential for its nucleolar localization, but were dispensable for the LLPS of NOP53. Functionally, NOP53 silencing diminished tumor cell growth, and significantly sensitized colorectal cancer (CRC) cells to radiotherapy. Mechanically, NOP53 negatively regulated p53 pathway in CRC cells treated with or without radiation. Importantly, data from clinical samples confirmed a correlation between NOP53 expression and tumor radio-resistance. Together, these results indicate an important role of NOP53 in radio-resistance, and provide a potential target for tumor radio-sensitization.

DNA damage-induced paraspeckle formation enhances DNA repair and tumor radioresistance by recruiting ribosomal protein P0.

Paraspeckles are mammal-specific membraneless nuclear bodies that participate in various biological processes. NONO, a central paraspeckle component, has been shown to play pivotal roles in DNA double-strand breaks (DSB) repair, whereas its underlying mechanism needs to be further disclosed. Here, using co-immunoprecipitation and mass spectrum, we identified ribosomal protein P0 (RPLP0) as a DSB-induced NONO-binding protein; RPLP0 binds to the RRM1 and RRM2 domains of NONO. Similar to NONO, RPLP0 enhances non-homologous end joining-mediated DSB repair, which was ascribed to a ribosome-independent manner. Interestingly, paraspeckles were induced as early as 15 min after irradiation; it further recruited nuclear RPLP0 to enhance its interaction with NONO. Radiation-induced NONO/RPLP0 complex subsequently anchored at the damaged DNA and increased the autophosphorylation of DNA-PK at Thr2609, thereby enhancing DSB repair. Consistently, in vivo and in vitro experiments showed that depletion of NONO sensitizes tumor cells to radiation. For patients with locally advanced rectal cancer, NONO expression was remarkably increased in tumor tissues and correlated with a poor response to radiochemotherapy. Our findings suggest a pivotal role of radiation-induced paraspeckles in DNA repair and tumor radioresistance, and provide a new insight into the ribosome-independent function of ribosomal proteins.

A 25-gene panel predicting the benefits of immunotherapy in head and neck squamous cell carcinoma.

Tumor mutation burden high (TMB-H) is widely used in the guidance of immune checkpoint blocking (ICB) therapy for head and neck squamous cell carcinoma (HNSCC) patients. However, a few patients still had a poor response. Therefore, it is necessary to investigate a better model to guide ICB therapy. We constructed a genomic mutation model conducive to ICB therapy using an available HNSCC dataset. Moreover, treatment procedures for patients with HNSCC from our internal cohort confirmed this model. Here, a genomic mutation signature based on a list of 25 candidate genes that are favorable for immunotherapy was established. Patients with combined mutation had a respectable clinical outcome under ICB treatment. Notably, compared with patients who obtained TMB-H (TMB ≥ 10, but did not have combined mutation), those patients with TMB-L (TMB < 10) and combined mutation acquired remarkably beneficial overall survival. Moreover, the combined mutation signature predicting the survival status of patients was superior to TMB, with a Youden index of 0.55. Furthermore, higher immune cell infiltration levels, more active cancer-immunity cycle activities and immune response pathways were observed in patients with combined mutation. Finally, our internal cohort further confirmed that combined mutated patients can benefit from ICB therapy rather than any other patients.

MRNIP condensates promote DNA double-strand break sensing and end resection.

The rapid recognition of DNA double-strand breaks (DSBs) by the MRE11/RAD50/NBS1 (MRN) complex is critical for the initiation of DNA damage response and DSB end resection. Here, we show that MRN complex interacting protein (MRNIP) forms liquid-like condensates to promote homologous recombination-mediated DSB repair. The intrinsically disordered region is essential for MRNIP condensate formation. Mechanically, the MRN complex is compartmentalized and concentrated into MRNIP condensates in the nucleus. After DSB formation, MRNIP condensates move to the damaged DNA rapidly to accelerate the binding of DSB by the concentrated MRN complex, therefore inducing the autophosphorylation of ATM and subsequent activation of DNA damage response signaling. Meanwhile, MRNIP condensates-enhanced MRN complex loading further promotes DSB end resection. In addition, data from xenograft models and clinical samples confirm a correlation between MRNIP and radioresistance. Together, these results reveal an important role of MRNIP phase separation in DSB response and the MRN complex-mediated DSB end resection.

A Pipeline for Predicting the Treatment Response of Neoadjuvant Chemoradiotherapy for Locally Advanced Rectal Cancer Using Single MRI Modality: Combining Deep Segmentation Network and Radiomics Analysis Based on "Suspicious Region".

Patients with locally advanced rectal cancer (LARC) who achieve a pathologic complete response (pCR) after neoadjuvant chemoradiotherapy (nCRT) typically have a good prognosis. An early and accurate prediction of the treatment response, i.e., whether a patient achieves pCR, could significantly help doctors make tailored plans for LARC patients. This study proposes a pipeline of pCR prediction using a combination of deep learning and radiomics analysis. Taking into consideration missing pre-nCRT magnetic resonance imaging (MRI), as well as aiming to improve the efficiency for clinical application, the pipeline only included a post-nCRT T2-weighted (T2-w) MRI. Unlike other studies that attempted to carefully find the region of interest (ROI) using a pre-nCRT MRI as a reference, we placed the ROI on a "suspicious region", which is a continuous area that has a high possibility to contain a tumor or fibrosis as assessed by radiologists. A deep segmentation network, termed the two-stage rectum-aware U-Net (tsraU-Net), is designed to segment the ROI to substitute for a time-consuming manual delineation. This is followed by a radiomics analysis model based on the ROI to extract the hidden information and predict the pCR status. The data from a total of 275 patients were collected from two hospitals and partitioned into four datasets: Seg-T (N = 88) for training the tsraUNet, Rad-T (N = 107) for building the radiomics model, In-V (N = 46) for internal validation, and Ex-V (N = 34) for external validation. The proposed method achieved an area under the curve (AUC) of 0.829 (95% confidence interval [CI]: 0.821, 0.837) on In-V and 0.815 (95% CI, 0.801, 0.830) on Ex-V. The performance of the method was considerable and stable in two validation sets, indicating that the well-designed pipeline has the potential to be used in real clinical procedures.

NONO phase separation enhances DNA damage repair by accelerating nuclear EGFR-induced DNA-PK activation.

Radioresistance is one of the main causes of cancer treatment failure, which leads to relapse and inferior survival outcome of cancer patients. Liquid-liquid phase separation (LLPS) of proteins is known to be involved in various biological processes, whereas its role in the regulation of radiosensitivity remains largely unknown. In this study, we characterized NONO, an RNA/DNA binding protein with LLPS capacity, as an essential regulator of tumor radioresistance. In vitro assay showed that NONO involved in DNA repair via non-homologous end joining (NHEJ) manner. NONO knockout significantly reduced DNA damage repair and sensitized tumor cells to irradiation in vitro and in vivo. NONO overexpression was correlated with an inferior survival outcome in cancer patients. Mechanically, NONO was associated with nuclear EGFR (nEGFR). Both irradiation and EGF treatment induced nEGFR accumulation, thereby increased the association between NONO and nEGFR. However, NONO was not a substrate of EGFR kinase. Furthermore, NONO promoted DNA damage-induced DNA-PK phosphorylation at T2609 by enhancing the interaction between EGFR and DNA-PK. Importantly, NONO protein formed high concentration LLPS droplets in vitro, and recruited EGFR and DNA-PK. Disruption of NONO droplets with LLPS inhibitor significantly reduced the interaction between EGFR and DNA-PK, and suppressed DNA damage-induced phosphorylation of T2609-DNA-PK. Taken together, LLPS of NONO recruits nuclear EGFR and DNA-PK and enhances their interaction, further increases DNA damage-activated pT2609-DNA-PK and promotes NHEJ-mediated DNA repair, finally leads to tumor radioresistance. NONO phase separation-mediated radioresistance may serve as a novel molecular target to sensitize tumor cell to radiotherapy.

Glycosylation of Siglec15 promotes immunoescape and tumor growth.

Siglec15 is a recently characterized immunosuppressive transmembrane protein, which expresses in various types of solid tumors and promotes cancer development. Several studies reported that Siglec15 is a prognostic biomarker of cancer patients, and targeting Siglec15 may be a promising strategy for cancer therapy. However, the regulation of Siglec15 function remains unclear. Here we show that the immunosuppression activity of Siglec15 is largely modulated by N-glycosylation. Through mass spectrum and site mutation analysis, we identified that Siglec15 was extensively glycosylated at N172 (N173 for mouse) in cancer cells. Meanwhile, Siglec15 N172Q had a similar molecular weight with PNGase-F-treated Siglec15, suggesting N172 as the only one glycosylation residue. In xenograft model, glycosylation deficiency of Siglec15 reduced tumor growth in C57BL/6 mice, but had no impact in nude mice, indicating the requirement of N-glycosylation for immunosuppressive function of Siglec15. Furthermore, colorectal cancer patients with high Siglec15 expression had a poor response to neoadjuvant chemo-radiotherapy and short survival time. Interestingly, removal of N-glycosylation enhances the detection of Siglec15, which may be employed in the prediction of immunotherapy response. Together, our results disclose a pivotal role of glycosylated Siglec15 in tumor immune escape, which may be a therapeutic target for cancer immunotherapy.

PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer.

Arginine methylation is an important posttranslational modification catalyzed by protein arginine methyltransferases (PRMTs). However, the role of PRMTs in colorectal cancer (CRC) progression is not well understood. Here we report that non-POU domain-containing octamer-binding protein (NONO) is overexpressed in CRC tissue and is a potential marker for poor prognosis in CRC patients. NONO silencing resulted in decreased proliferation, migration, and invasion of CRC cells, whereas overexpression had the opposite effect. In a xenograft model, tumors derived from NONO-deficient CRC cells were smaller than those derived from wild-type (WT) cells, and PRMT1 inhibition blocked CRC xenograft progression. A mass spectrometry analysis indicated that NONO is a substrate of PRMT1. R251 of NONO was asymmetrically dimethylated by PRMT1 in vitro and in vivo. Compared to NONO WT cells, NONO R251K mutant-expressing CRC cells showed reduced proliferation, migration, and invasion, and PRMT1 knockdown or pharmacological inhibition abrogated the malignant phenotype associated with NONO asymmetric dimethylation in both KRAS WT and mutant CRC cells. Compared to adjacent normal tissue, PRMT1 was highly expressed in the CRC zone in clinical specimens, which was correlated with poor overall survival in patients with locally advanced CRC. These results demonstrate that PRMT1-mediated methylation of NONO at R251 promotes CRC growth and metastasis, and suggest that PRMT1 inhibition may be an effective therapeutic strategy for CRC treatment regardless of KRAS mutation status.

Impact of Cold Ischemic Time and Freeze-Thaw Cycles on RNA, DNA and Protein Quality in Colorectal Cancer Tissues Biobanking.

Tissue-derived RNA, DNA and protein samples become more and more crucial for molecular detection in clinical research, personalized and targeted cancer therapy. This study evaluated how to biobanking colorectal tissues through examining the influences of cold ischemic time and freeze-thaw cycles on RNA, DNA and protein integrity. Here, 144 pairs of tumor and normal colorectal tissues were used to investigate the impact of cold ischemic times (0-48h) on RNA, DNA and protein integrity at on ice or room temperature conditions. Additionally, 45 pairs of tissues experienced 0-9 freeze-thaw cycles, and then the RNA, DNA and protein quality were analyzed. On ice, RNA, DNA and protein from colorectal tumor and normal tissues were all stable up to 48h after surgery. At room temperature, RNA in colorectal tumor and normal tissues began to degrade at 8h and 24h, respectively. Meanwhile, the tumor tissues DNA degradation occurred at 24h after surgery at room temperature. Similarly, the protein expression level of tumor and normal tissues began to change at 24h after the surgery at room temperature. Interestingly, tissue RNA and DNA remained stable even after 9 freeze-thaw cycles, whereas the proteins levels were remarkably changed after 7 freeze-thaw cycles. This study provided a useful evidence on how to store human colorectal tissues for biobanking. Preserving the surgical colorectal tissue on ice was an effective way to prevent RNA, DNA and protein degradation. Importantly, more than 7 repeated freeze-thaw cycles were not recommended for colorectal tissues.