RNA-binding protein HuR reprograms immune T cells and promotes oral squamous cell carcinoma.

Hu Antigen R, also known as ELAVL1 (HuR), is a key posttranscriptional regulator in eukaryotic cells. HuR overexpression promotes several malignancies, including head and neck squamous cell carcinoma (HNSCC). However, its immune dysfunction-associated tumorigenesis pathways remain unknown. We examined HuR's effects on oral malignancies and immune cell function in vitro and in vivo using oral carcinoma cells and transgenic HuR knockout (KO) mice. CRISPR/Cas9-mediated HuR deletion in mice syngeneic oral cancer cells eliminated colony formation and tumor development. HuR-KO tumors had a lower tumor volume, fewer CD4+CD25+FoxP3+ regulatory T cells, and more CD8+ T cells, suggesting that HuR may suppress the immune response during oral cancer progression. In contrast, HuR KO oral epithelial tissues are resistant to 4NQO-induced oral malignancies compared to control tumor-bearing mice. HuR KO mice showed fewer Tregs and greater IFN levels than WT tumor-bearing mice, suggesting anticancer activity. Finally, the HuR inhibitor pyrvinium pamoate lowers tumor burden by enhancing CD8+ infiltration at the expense of CD4+, suggesting anticancer benefits. Thus, HuR-dependent oral neoplasia relies on immunological dysfunction, suggesting that decreasing HuR may boost antitumor potential and offer a novel HNSCC therapy.

Sphingosine-1-Phosphate Inhibition Increases Endoplasmic Reticulum Stress to Enhance Oxaliplatin Sensitivity in Pancreatic Cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer resistant to current therapies, including oxaliplatin (Oxa). Growing evidence supports the ability of cancers to harness sphingolipid metabolism for survival. Sphingosine-1-phosphate (S1P) is an anti-apoptotic, pro-survival mediator that can influence cellular functions such as endoplasmic reticulum (ER) stress. We hypothesize that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress to improve therapeutic response to Oxa in PDAC. Methods: RNA sequencing data of sphingolipid mediators from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) datasets were analyzed. Murine and human PDAC cell lines were treated with small interfering RNA (siRNA) against sphingosine kinase-2 (SPHK2) or ABC294640 (ABC) and incubated with combinations of vehicle control or Oxa. In an orthotopic syngeneic KPC PDAC model, tumors were treated with either vehicle control, Oxa, ABC, or combination therapy. Results: RNA sequencing analysis revealed multiple significantly differentially expressed sphingolipid mediators (P < 0.05). In vitro, both siRNA knockdown of SPHK2 and ABC sensitized cells to Oxa therapy (P < 0.05), and induced eukaryotic initiation factor 2α (eIF2α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation, hallmarks of ER stress. In vitro therapy also increased extracellular high mobility group box 1 (HMGB1) release (P < 0.05), necessary for immunogenic cell death (ICD). In vivo combination therapy increased apoptotic markers as well as the intensity of HMGB1 staining compared to control (P < 0.05). Conclusions: Our evidence suggests that sphingolipid metabolism is dysregulated in PDAC. Furthermore, S1P inhibition can sensitize PDAC to Oxa therapy through increasing ER stress and can potentiate ICD induction. This highlights a potential therapeutic target for chemosensitizing PDAC as well as an adjunct for future chemoimmunotherapy strategies.

Critical evaluation of an autologous peripheral blood mononuclear cell-based humanized cancer model.

The use of humanized mouse models for oncology is rapidly expanding. Autologous patient-derived systems are particularly attractive as they can model the human cancer's heterogeneity and immune microenvironment. In this study, we developed an autologous humanized mouse cancer model by engrafting NSG mice with patient-derived xenografts and infused matched peripheral blood mononuclear cells (PBMCs). We first defined the time course of xenogeneic graft-versus-host-disease (xGVHD) and determined that only minimal xGVHD was observed for up to 8 weeks. Next, colorectal and pancreatic cancer patient-derived xenograft bearing NSG mice were infused with 5x106 human PBMCS for development of the humanized cancer models (iPDX). Early after infusion of human PBMCs, iPDX mice demonstrated engraftment of human CD4+ and CD8+ T cells in the blood of both colorectal and pancreatic cancer patient-derived models that persisted for up to 8 weeks. At the end of the experiment, iPDX xenografts maintained the features of the primary human tumor including tumor grade and cell type. The iPDX tumors demonstrated infiltration of human CD3+ cells with high PD-1 expression although we observed significant intra and inter- model variability. In summary, the iPDX models reproduced key features of the corresponding human tumor. The observed variability and high PD-1 expression are important considerations that need to be addressed in order to develop a reproducible model system.

Loss of CPAP causes sustained EGFR signaling and epithelial-mesenchymal transition in oral cancer.

Higher epidermal growth factor receptor (EGFR) signaling can contribute to tumor metastasis and resistance to therapies in oral squamous cell carcinoma (OSCC). EGFR signaling can promote epithelial-mesenchymal transition (EMT) in OSCC. EMT is a process by which epithelial cells acquire invasive properties and it can contribute to tumor metastasis. Not only do the abnormal functions of microtubule and microtubule-organizing centers (MTOC) such as centrosomes lead to cancers, but also the malignant tissues are characterized by aberrant centriolar features and amplified centrosomes. Microtubule inhibition therapies increase the sensitivity to EGFR targeting drugs in various cancers. In this study, we show that the loss of expression of a microtubule/tubulin binding protein, centrosomal protein 4.1-associated protein (CPAP), which is critical for centriole biogenesis and normal functioning of the centrosome, caused an increase in the EGFR levels and its signaling and, enhanced the EMT features and invasiveness of OSCC cells. Further, depletion of CPAP enhanced the tumorigenicity of these cells in a xeno-transplant model. Importantly, CPAP loss-associated EMT features and invasiveness of multiple OSCC cells were attenuated upon depletion of EGFR in them. On the other hand, we found that CPAP protein levels were higher in EGF treated OSCC cells as well as in oral cancer tissues, suggesting that the frequently reported aberrant centriolar features of tumors are potentially a consequence, but not the cause, of tumor progression. Overall, our novel observations show that, in addition to its known indispensable role in centrosome biogenesis, CPAP also plays a vital role in suppressing tumorigenesis in OSCC by facilitating EGFR homeostasis.

High-Fat Diet Drives an Aggressive Pancreatic Cancer Phenotype.

BACKGROUND: Emerging evidence indicates associations between high-fat diet (HFD), metabolic syndrome (MetS), and increased risk of pancreatic cancer. However, individual components of an HFD that increase cancer risk have not been isolated. In addition, a specific pattern of cytokine elevation by which MetS drives pancreatic tumor progression is not well described. We hypothesized that oleic acid (OA), a major component of HFD, would augment pancreatic neoplastic processes. METHODS: An orthotopic pancreatic cancer model with Panc02 cells was used to compare the effect of low-fat diet to OA-based HFD on cancer progression. Tumors were quantitated, analyzed by immunohistochemistry. In addition, serum cytokine levels were quantitated. Proliferation, migration assays, and expression of epithelial-to-mesenchymal transition factors were evaluated on Panc02 and MiaPaCa-2 pancreatic cancer cells cultured in high concentrations of OA. RESULTS: HFD tumor-bearing mice (n = 8) had an 18% weight increase (P < 0.001) and increased tumor burden (P < 0.05) compared with the low-fat diet tumor-bearing group (n = 6). HFD tumors had significantly increased angiogenesis (P < 0.001) and decreased apoptosis (P < 0.05). Serum of HFD mice demonstrated increased levels of glucagon and glucagon-like peptide-1. Two pancreatic cancer cell lines cultured in OA demonstrated significant increases in proliferation (P < 0.001) and a >2.5-fold increase in cell migration (P < 0.001) when treated with OA. Panc02 treated with OA had increased expression of epithelial-to-mesenchymal transition factors SNAI-1 (Snail) and Zeb-1(P < 0.01). CONCLUSIONS: High-fat conditions in vitro and in vivo resulted in an aggressive pancreatic cancer phenotype. Our data support further investigations elucidating molecular pathways augmented by MetS conditions to identify novel therapeutic strategies for pancreatic cancer.

Modeling rectal cancer to advance neoadjuvant precision therapy.

Progress in rectal cancer therapy has been hindered by the lack of effective disease-specific preclinical models that account for the unique molecular profile and biology of rectal cancer. Thus, we developed complementary patient-derived xenograft (PDX) and subsequent in vitro tumor organoid (PDTO) platforms established from preneoadjuvant therapy rectal cancer specimens to advance personalized care for rectal cancer patients. Multiple endoscopic samples were obtained from 26 Stages 2 and 3 rectal cancer patients prior to receiving 5FU/RT and implanted subcutaneously into NSG mice to generate 15 subcutaneous PDXs. Second passaged xenografts demonstrated 100% correlation with the corresponding human cancer histology with maintained mutational profiles. Individual rectal cancer PDXs reproduced the 5FU/RT response observed in the corresponding human cancers. Similarly, rectal cancer PDTOs reproduced significant heterogeneity in cellular morphology and architecture. PDTO in vitro 5FU/RT treatment response replicated the clinical 5FU/RT neoadjuvant therapy pathologic response observed in the corresponding patient tumors (p < 0.05). The addition of cetuximab to the 5FU/RT regiment was significantly more sensitive in the rectal cancer PDX and PDTOs with wild-type KRAS compared to mutated KRAS (p < 0.05). Considering the close relationship between the patient's cancer and the corresponding PDX/PDTO, rectal cancer patient-derived research platforms represent powerful translational research resources as population-based tools for biomarker discovery and experimental therapy testing. In addition, our findings suggest that cetuximab may enhance RT effectiveness by improved patient selection based on mutational profile in addition to KRAS or by developing a protocol using PDTOs to identify sensitive patients.

The Long (lncRNA) and Short (miRNA) of It: TGFß-Mediated Control of RNA-Binding Proteins and Noncoding RNAs.

RNA-binding proteins (RBP) and noncoding RNAs (ncRNA), such as long noncoding RNAs (lncRNA) and microRNAs (miRNA), control co- and posttranscriptional gene regulation (PTR). At the PTR level, RBPs and ncRNAs contribute to pre-mRNA processing, mRNA maturation, transport, localization, turnover, and translation. Deregulation of RBPs and ncRNAs promotes the onset of cancer progression and metastasis. Both RBPs and ncRNAs are altered by signaling cascades to cooperate or compete with each other to bind their nucleic acid targets. Most importantly, transforming growth factor-beta (TGFß) signaling plays a significant role in controlling gene expression patterns by targeting RBPs and ncRNAs. Because of TGFß signaling in cancer, RBP-RNA or RNA-RNA interactions are altered and cause enhanced cell growth and tumor cell dissemination. This review focuses on the emerging concepts of TGFß signaling on posttranscriptional gene regulation and highlights the implications of RBPs and ncRNAs in cancer progression and metastasis. Mol Cancer Res; 16(4); 567-79. ©2018 AACR.

Lack of p53 Augments Antitumor Functions in Cytolytic T Cells.

Repetitive stimulation of T-cell receptor (TCR) with cognate antigen results in robust proliferation and expansion of the T cells, and also imprints them with replicative senescence signatures. Our previous studies have shown that life-span and antitumor function of T cells can be enhanced by inhibiting reactive oxygen species (ROS) or intervening with ROS-dependent JNK activation that leads to its activation-induced cell death. Because tumor suppressor protein p53 is also a redox active transcription factor that regulates cellular ROS generation that triggers downstream factor-mediating apoptosis, we determined if p53 levels could influence persistence and function of tumor-reactive T cells. Using h3T TCR transgenic mice, with human tyrosinase epitope-reactive T cells developed on p53 knockout (KO) background, we determined its role in regulating antitumor T-cell function. Our data show that as compared with h3T cells, h3T-p53 KO T cells exhibited enhanced glycolytic commitment that correlated with increased proliferation, IFNγ secretion, cytolytic capacity, expression of stemness gene signature, and decreased TGF-ß signaling. This increased effector function correlated to the improved control of subcutaneously established murine melanoma after adoptive transfer of p53-KO T cells. Pharmacological inhibition of human TCR-transduced T cells using a combination of p53 inhibitors also potentiated the T-cell effector function and improved persistence. Thus, our data highlight the key role of p53 in regulating the tumor-reactive T-cell response and that targeting this pathway could have potential translational significance in adoptive T-cell therapy. Cancer Res; 76(18); 5229-40. ©2016 AACR.

Mel-18 controls the enrichment of tumor-initiating cells in SP fraction in mouse breast cancer.

Side population (SP) cell analysis has been used to identify and isolate a minor population of cells with stem cell properties in normal tissues and in many cancers including breast cancer cells. However, the molecular mechanisms that operate in tumor-initiating cells (TICs) in SP fraction remain unclear. The Polycomb group genes, including Bmi1 and Mel-18, have been implicated in the maintenance of hematopoietic stem cells (HSCs) and suggested to be oncogenic and tumor suppressive, respectively, in breast cancer. In this study, we determined the critical role of Mel-18 in the enrichment mechanisms of TICs with the SP phenotype in a mouse breast cancer cell line, MMK3, that was established from a breast cancer developed spontaneously in Mel-18+/- mice. The Mel-18 protein expression level significantly correlates to the percentage of SP fraction in the mouse breast cancer cell line MMK3 series. The comparison between MMK3V3 (V3) cells containing one copy of the Mel-18 gene and MMK3S2 (S2) cells having twice the amount of Mel-18 expression clearly demonstrates the above relationship. Similar results obtained with the percentage of ALDH+ cells in V3 and S2 further confirmed the correlation between protein expression level of Mel-18 and the TICs. More importantly, transplantation of SP and non-SP cells of V3 and S2 cells into the NOD/SCID mice clearly showed that the heterozygous level of Mel-18 leads to the disappearance of enrichment of TICs into SP fraction in vivo. Stem cell pathway focused gene expression profiling of V3 and S2 cells revealed that the genes Abcg2, Aldh1a1 and Dhh were highly down-regulated in V3 compared to S2. These results indicate that the precise Mel-18 expression level controls TIC enrichment mechanisms through the regulation of channel molecule of Abcg2 and functional TIC marker of Aldhlal. In conclusion, our findings revealed the significance of fine-tuning mechanisms for Mel-18 protein expression level in the maintenance of TIC into SP fractions in mouse breast cancer.

Epigenetic regulator polycomb group protein complexes control cell fate and cancer.

The chromatin-associated Polycomb group (PcG) proteins were first identified in genetic screens for homeotic transformations in Drosophila melanogaster. Besides body patterning, members of the PcG are now known to regulate epigenetic cellular memory, stem cell self-renewal, and cancer development. Here, we discuss the multifarious functions of the PcG family, isoforms of protein complexes, and its enzymatic activities, for example histone methylation, links to DNA methylation, its phosphorylation status, H2A mono-ubiquitination, SUMOylation, and links to non-coding RNA. We also discuss the function of cytosolic PcG complexes as a regulator of receptor-induced actin polymerization and proliferation in a methylation-dependent manner. We propose that the functional versatility of PcG protein complexes contributed significantly to the complexity of heritable gene repression mechanisms, signal transduction, and cell proliferation in cancer development.