The Functions of PCNA in Tumor Stemness and Invasion.

Invasion is the most prominent lethal feature of malignant cancer. However, how cell proliferation, another important feature of tumor development, is integrated with tumor invasion and the subsequent cell dissemination from primary tumors is not well understood. Proliferating cell nuclear antigen (PCNA) is essential for DNA replication in cancer cells. Loss of phosphorylation at tyrosine 211 (Y211) in PCNA (pY211-PCNA) mitigates PCNA function in proliferation, triggers replication fork arrest/collapse, which in turn sets off an anti-tumor inflammatory response, and suppresses distant metastasis. Here, we show that pY211-PCNA is important in stromal activation in tumor tissues. Loss of the phosphorylation resulted in reduced expression of mesenchymal proteins as well as tumor progenitor markers, and of the ability of invasion. Spontaneous mammary tumors that developed in mice lacking Y211 phosphorylation contained fewer tumor-initiating cells compared to tumors in wild-type mice. Our study demonstrates a novel function of PCNA as an essential factor for maintaining cancer stemness through Y211 phosphorylation.

miR-4759 suppresses breast cancer through immune checkpoint blockade.

Programmed cell death protein 1 (PD-1)/ programmed cell death protein ligand 1 (PD-L1) is the key immune checkpoint governing evasion of advanced cancer from immune surveillance. Immuno-oncology (IO) therapy targeting PD-1/PD-L1 with traditional antibodies is a promising approach to multiple cancer types but to which the response rate remains moderate in breast cancer, calling for the need of exploring alternative IO targeting approaches. A miRNA-gene network was integrated by a bioinformatics approach and corroborated with The Cancer Genome Atlas (TCGA) to screen miRNAs regulating immune checkpoint genes and associated with patient survival. Here we show the identification of a novel microRNA miR-4759 which repressed RNA expression of the PD-L1 gene. miR-4759 targeted the PD-L1 gene through two binding motifs in the 3' untranslated region (3'-UTR) of PD-L1. Reconstitution of miR-4759 inhibited PD-L1 expression and sensitized breast cancer cells to killing by immune cells. Treatment with miR-4759 suppressed tumor growth of orthotopic xenografts and promoted tumor infiltration of CD8+ T lymphocytes in immunocompetent mice. In contrast, miR-4759 had no effect to tumor growth in immunodeficient mice. In patients with breast cancer, expression of miR-4759 was preferentially downregulated in tumors compared to normal tissues and was associated with poor overall survival. Together, our results demonstrated miR-4759 as a novel non-coding RNA which promotes anti-tumor immunity of breast cancer.

Suppression of breast cancer cells resistant to a pure anti-estrogen with CAR-transduced natural killer cells.

Anti-estrogens as hormone therapy are the mainstay treatment for estrogen receptor (ER)-positive breast cancer. ER inhibitors through modulating the transcriptional function of ER have been the frontline anti-estrogens to which refractory phenotype often developed in advanced cancer. The anti-estrogen fulvestrant is currently the only clinically approved pure anti-estrogen which causes ER degradation. However, resistance to fulvestrant still occurs and unfortunately it leaves few choices other than chemotherapy as the later-line treatments to fulvestrant-resistant tumors. Here we show that fulvestrant resistance was accompanied by increased expression of a number of innate immune response genes including the natural killer (NK) cell ligand B7-H6 on the cell surface. In an attempt to overcome the drug resistance phenotype, a NK-based molecular approach taking advantage of a chimeric antigen receptor (CAR) system targeting B7-H6 was established and tested in cells with acquired resistance to fulvestrant. The results demonstrate that the cell therapy approach as a single agent can effectively induce cell death of the resistant cancer cells which is enhanced by the increased expression of cell surface B7-H6. This approach departs from the traditional strategies of conquering anti-estrogen resistant breast cancer and offers a new avenue to eradicate hormone-refractory malignant solid tumors.

Evading immune surveillance via tyrosine phosphorylation of nuclear PCNA.

Increased DNA replication and metastasis are hallmarks of cancer progression, while deregulated proliferation often triggers sustained replication stresses in cancer cells. How cancer cells overcome the growth stress and proceed to metastasis remains largely elusive. Proliferating cell nuclear antigen (PCNA) is an indispensable component of the DNA replication machinery. Here, we show that phosphorylation of PCNA on tyrosine 211 (pY211-PCNA) regulates DNA metabolism and tumor microenvironment. Abrogation of pY211-PCNA blocks fork processivity, resulting in biogenesis of single-stranded DNA (ssDNA) through a MRE11-dependent mechanism. The cytosolic ssDNA subsequently induces inflammatory cytokines through a cyclic GMP-AMP synthetase (cGAS)-dependent cascade, triggering an anti-tumor immunity by natural killer (NK) cells to suppress distant metastasis. Expression of pY211-PCNA is inversely correlated with cytosolic ssDNA and associated with poor survival in patients with cancer. Our results pave the way to biomarkers and therapies exploiting immune responsiveness to target metastatic cancer.

Imatinib (STI571) Inhibits the Expression of Angiotensin-Converting Enzyme 2 and Cell Entry of the SARS-CoV-2-Derived Pseudotyped Viral Particles.

A group of clinically approved cancer therapeutic tyrosine kinase inhibitors was screened to test their effects on the expression of angiotensin-converting enzyme 2 (ACE2), the cell surface receptor for SARS-CoV-2. Here, we show that the receptor tyrosine kinase inhibitor imatinib (also known as STI571, Gleevec) can inhibit the expression of the endogenous ACE2 gene at both the transcript and protein levels. Treatment with imatinib resulted in inhibition of cell entry of the viral pseudoparticles (Vpps) in cell culture. In FVB mice orally fed imatinib, tissue expression of ACE2 was reduced, specifically in the lungs and renal tubules, but not in the parenchyma of other organs such as the heart and intestine. Our finding suggests that receptor tyrosine kinases play a role in COVID-19 infection and can be therapeutic targets with combined treatments of the best conventional care of COVID-19.

Long non-coding RNA HOTAIR in circulatory exosomes is correlated with ErbB2/HER2 positivity in breast cancer.

Cancer cells are known to produce and secret extracellular vesicles for intercellular communication through the carried cargos. HOTAIR (HOX transcript antisense intergenic RNA), a well-studied long non-coding RNA (lncRNA), plays a critical role in cancer progression. In several cancer types it has been shown that HOTAIR-containing exosomes are produced by cancer cells. Here we show that circulatory exosomal HOTAIR is present in breast cancer patients and explores the pathological correlation with the disease. Exosomes were isolated by matrix-based precipitation from conditioned media of cultured breast cancer cell lines as well as blood samples of recently recruited breast cancer patients. HOTAIR RNA in exosomes was detected by quantitative reverse transcriptase-mediated polymerase chain reaction (qRT-PCR). Expression of exosomal HOTAIR was positively correlated with status of the receptor tyrosine kinase (RTK) ErbB2 (also known as HER2/neu) in tumor tissues. The causal correlation of ErbB2 and HOTAIR was validated in isogenic breast cancer cell lines with and without ectopic ErbB2 expression. Our finding provides a molecular basis to develop novel liquid biopsy biomarkers and targeted therapies with improved precision for malignant breast cancer.

The exosomal compartment protects epidermal growth factor receptor from small molecule inhibitors.

Epidermal growth factor receptor (EGFR) plays a significant role in promoting breast cancer progression. However, targeting EGFR as a single treatment only resulted in moderate efficacy to the disease. The underlying mechanism of low responsiveness to EGFR inhibition remains largely unclear. Tumor-secreted extracellular vesicles (EVs) play a crucial role in mediating intercellular communication between tumor and stromal cells in local microenvironment and distant metastatic niche. Extracellular vesicles mediate cell-to-cell transfer of lipids, nucleic acids, and proteins. Although numerous recent studies have demonstrated exchanges of extracellular vesicles between cancer cells and the recipient cells contribute to tumor proliferation, invasion, and metastasis, yet little is known how the exosomal compartment responds to targeted therapies and their role in promoting drug resistance. In the current study we used a triple-negative breast cancer model to show that EV-encapsulated EGFR is protected from targeted inhibitors of EGFR and can trigger signaling pathway in recipient cancer cells, promoting proliferation and migration ability in vitro. Taken together, our study suggested a novel mechanism of drug resistance entailing the EV compartment, such as exosomes, as a target shelter which when released can signal for tumor promotion in the recipient cancer cells.

Arecoline N-oxide regulates oral squamous cell carcinoma development through NOTCH1 and FAT1 expressions.

Areca nut has been evaluated as a group I carcinogen to humans. However, the exact compounds of areca nut causing oral cancer remain unproven. Previous findings from our lab revealed that arecoline N-oxide (ANO), a metabolite of arecoline, exhibits an oral fibrotic effect in immune-deficient NOD/SCID mice. The aim of this study is to investigate the oral potentially malignant disorders (OPMD) inductive activity between areca-alkaloid arecoline and its metabolite ANO in C57BL/6 mice. Our findings show that ANO showed higher activity in inducing hyperplasia with leukoplakia and collagen deposition in C57BL/6 mice compared with the arecoline treated groups. Importantly, immunohistochemical studies showed significant upregulation of NOTCH1, HES1, FAT1, PCNA, and Ki67 expressions in the pathological hyperplastic part. In addition, in vitro studies showed that upregulation of NOTCH1 and FAT1 expressions in ANO treated HGF-1 and DOK cell models. We found that NOTCH1 regulates TP53 expression from NOTCH1 knockdown oral cancer cells. The DNA damage was significantly increased after arecoline and ANO treatment. Further, we found that arecoline-induced H2AX expression was regulated by FMO3. Altogether, our findings show that ANO exhibited higher toxicity in OPMD activity and play a significant role in the induction of areca nut mediated oral tumorigenesis.

Arecoline N-Oxide Upregulates Caspase-8 Expression in Oral Hyperplastic Lesions of Mice.

Areca nut is strongly associated with oral squamous cell carcinoma (OSCC) occurrence. Arecoline N-oxide (ANO), a metabolite of the areca alkaloid arecoline, exhibits an oral fibrotic effect in NOD/SCID mice. Caspase-8, a cysteine protease encoded by the CASP8 gene, is a central mediator in the extrinsic apoptotic pathway via death receptors. Deregulation of caspase-8 in OSCC has been reported. This study investigates the regulation of caspase-8 in ANO-induced oral squamous epithelial hyperplasia that represents the initial highly proliferative stage of oral carcinogenesis. CASP8 somatic mutations were identified from whole-exome sequencing of OSCC samples. Immunohistochemical staining showed upregulation of caspase-8 in ANO-induced hyperplasia of both NOD-SCID and C57BL/6 mice. Levels of expression of CASP8, APAF-1, BAX, and BAD increased in ANO-treated DOK cells. Co-localization of increased caspase-8 and PCNA levels was detected in ANO-induced hyperplastic lesions, whereas no co-localization among γ-H2A.X, caspase-3, and upregulated caspase-8 was observed. The findings indicate that upregulation of caspase-8 is involved in cell proliferation rather than apoptosis during the initial stage of ANO-mediated oral tumorigenesis.

Somatic Mutations and Genetic Variants of NOTCH1 in Head and Neck Squamous Cell Carcinoma Occurrence and Development.

A number of genetic variants have been associated with cancer occurrence, however it may be the acquired somatic mutations (SMs) that drive cancer development. This study investigates the potential SMs and related genetic variants associated with the occurrence and development of head and neck squamous cell carcinoma (HNSCC). We identified several SMs in NOTCH1 from whole-exome sequencing and validated them in a 13-year cohort of 128 HNSCC patients using a high-resolution melting analysis and resequencing. Patients who have NOTCH1 SMs show higher 5-year relapse-free recurrence (P = 0.0013) and lower survival proportion (P = 0.0447) when the risk-associated SMs were analysed by Cox proportional hazard models. Interestingly, the NOTCH1 gene rs139994842 that shares linkage with SMs is associated with HNSCC risk (OR = 3.46), increasing when SMs in NOTCH1 are involved (OR = 7.74), and furthermore when there are SMs in conjunction to betel quid chewing (OR = 32.11), which is a related independent environmental risk factor after adjusting for substances use (alcohol, betel quid, cigarettes) and age. The findings indicate that betel quid chewing is highly associated with NOTCH1 SMs (especially with changes in EGF-like domains), and that rs139994842 may potentially serve as an early predictive and prognostic biomarker for the occurrence and development of HNSCC.