Coordinated translational control of multiple immune checkpoints by the integrated stress response pathway in lung cancer.

Unlabelled: The integrated stress response (ISR) is an adaptive pathway hijacked by cancer cells to survive cellular stresses in the tumor microenvironment. ISR activation potently induces Programmed Death Ligand 1 (PD-L1), leading to suppression of anti-tumor immunity. Here we sought to uncover additional immune checkpoint proteins regulated by the ISR to elucidate mechanisms of tumor immune escape.

Capturing heterogeneity in PDX models: representation matters.

Patient derived tumor xenografts (PDXs) are important models for pre-clinical testing in cancer research and personalized medicine. PDXs often represent patient tumors with high similarity in terms of histology and driver mutations. However, certain limitations exist that warrant a detailed understanding of PDX heterogeneity and evolution.

Deep Learning-Based H-Score Quantification of Immunohistochemistry-Stained Images.

Immunohistochemistry (IHC) is a well-established and commonly used staining method for clinical diagnosis and biomedical research. In most IHC images, the target protein is conjugated with a specific antibody and stained using diaminobenzidine (DAB), resulting in a brown coloration, whereas hematoxylin serves as a blue counterstain for cell nuclei. The protein expression level is quantified through the H-score, calculated from DAB staining intensity within the target cell region.

Translational Control of Immune Evasion in Cancer.

Mechanisms that control translation play important roles in tumor progression and metastasis. Emerging evidence has revealed that dysregulated translation also impacts immune evasion in response to cellular or oncogenic stress. Here, we summarize current knowledge regarding the translational control of immune checkpoints and implications for cancer immunotherapies.

eIF5B drives integrated stress response-dependent translation of PD-L1 in lung cancer.

Cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T cells, allowing tumors to suppress T cell activity. Clinical trials utilizing antibodies that disrupt the PD-1/PD-L1 checkpoint have yielded remarkable results, with anti-PD-1 immunotherapy approved as first-line therapy for lung cancer patients. We used CRISPR-based screening to identify regulators of PD-L1 in human lung cancer cells, revealing potent induction of PD-L1 upon disruption of heme biosynthesis.

Ex Vivo Transposon-Mediated Genetic Screens for Cancer Gene Discovery.

Transposon mutagenesis has emerged as a powerful methodology for functionally annotating cancer genomes. Although in vivo transposon-mediated forward genetic screens have proven to be valuable for cancer gene identification, they are also time consuming and resource intensive. To facilitate the rapid and cost-effective identification of genes that regulate tumor-promoting pathways, we developed a complementary ex vivo transposon mutagenesis approach wherein human or mouse cells growing in culture are mutagenized and screened for the acquisition of specific phenotypes in vitro or in vivo, such as growth factor independence or tumor-forming ability.

Modulation of Mutant -Driven Lung Tumorigenesis by Gain or Loss of PCDH7 Function.

PROTOCADHERIN 7 (PCDH7), a transmembrane receptor and member of the Cadherin superfamily, is frequently overexpressed in lung adenocarcinoma and is associated with poor clinical outcome. Although PCDH7 was recently shown to promote transformation and facilitate brain metastasis in lung and breast cancers, decreased PCDH7 expression has also been documented in colorectal, gastric, and invasive bladder cancers. These data suggest context-dependent functions for PCDH7 in distinct tumor types.

Correction: SRC-2-mediated coactivation of anti-tumorigenic target genes suppresses MYC-induced liver cancer.

[This corrects the article DOI: 10.1371/journal.pgen.

Advances in functional genetic screening with transposons and CRISPR/Cas9 to illuminate cancer biology.

Large-scale genome sequencing studies have identified a wealth of mutations in human tumors and have dramatically advanced the field of cancer genetics. However, the functional consequences of an altered gene in tumor progression cannot always be inferred from mutation status alone. This underscores the critical need for complementary methods to assign functional significance to mutated genes in cancer.

A Conserved Splicing Silencer Dynamically Regulates O-GlcNAc Transferase Intron Retention and O-GlcNAc Homeostasis.

Modification of nucleocytoplasmic proteins with O-GlcNAc regulates a wide variety of cellular processes and has been linked to human diseases. The enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) add and remove O-GlcNAc, but the mechanisms regulating their expression remain unclear. Here, we demonstrate that retention of the fourth intron of OGT is regulated in response to O-GlcNAc levels.

SRC-2-mediated coactivation of anti-tumorigenic target genes suppresses MYC-induced liver cancer.

Hepatocellular carcinoma (HCC) is the fifth most common solid tumor in the world and the third leading cause of cancer-associated deaths. A Sleeping Beauty-mediated transposon mutagenesis screen previously identified mutations that cooperate with MYC to accelerate liver tumorigenesis. This revealed a tumor suppressor role for Steroid Receptor Coactivator 2/Nuclear Receptor Coactivator 2 (Src-2/Ncoa2) in liver cancer.

PROTOCADHERIN 7 Acts through SET and PP2A to Potentiate MAPK Signaling by EGFR and KRAS during Lung Tumorigenesis.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-associated deaths worldwide. Given the efficacy of membrane proteins as therapeutic targets in human malignancies, we examined cell-surface receptors that may act as drivers of lung tumorigenesis. Here, we report that the PROTOCADHERIN PCDH7 is overexpressed frequently in NSCLC tumors where this event is associated with poor clinical outcome.

Hepatoblastoma modeling in mice places Nrf2 within a cancer field established by mutant -catenin.

Aberrant wnt/β-catenin signaling and amplification/overexpression of Myc are associated with hepatoblastoma (HB), the most prevalent type of childhood liver cancer. To address their roles in the pathogenesis of HB, we generated mice in which Myc and mutant β-catenin were targeted to immature cells of the developing mouse liver. Perinatal coexpression of both genes promoted the preferential development of HBs over other tumor types in neonatal mice, all of which bore striking resemblance to their human counterparts.

Comprehensive Ex Vivo Transposon Mutagenesis Identifies Genes That Promote Growth Factor Independence and Leukemogenesis.

Aberrant signaling through cytokine receptors and their downstream signaling pathways is a major oncogenic mechanism underlying hematopoietic malignancies. To better understand how these pathways become pathologically activated and to potentially identify new drivers of hematopoietic cancers, we developed a high-throughput functional screening approach using ex vivo mutagenesis with the Sleeping Beauty transposon. We analyzed over 1,100 transposon-mutagenized pools of Ba/F3 cells, an IL3-dependent pro-B-cell line, which acquired cytokine independence and tumor-forming ability.

A role for retrotransposon LINE-1 in fetal oocyte attrition in mice.

Fetal oocyte attrition (FOA) is a conserved but poorly understood process of elimination of more than two-thirds of meiotic prophase I (MPI) oocytes before birth. We now implicate retrotransposons LINE-1 (L1), activated during epigenetic reprogramming of the embryonic germline, in FOA in mice. We show that wild-type fetal oocytes possess differential nuclear levels of L1ORF1p, an L1-encoded protein essential for L1 ribonucleoprotein particle (L1RNP) formation and L1 retrotransposition.

Controlled insertional mutagenesis using a LINE-1 (ORFeus) gene-trap mouse model.

A codon-optimized mouse LINE-1 element, ORFeus, exhibits dramatically higher retrotransposition frequencies compared with its native long interspersed element 1 counterpart. To establish a retrotransposon-mediated mouse model with regulatable and potent mutagenic capabilities, we generated a tetracycline (tet)-regulated ORFeus element harboring a gene-trap cassette. Here, we show that mice expressing tet-ORFeus broadly exhibit robust retrotransposition in somatic tissues when treated with doxycycline.

Poly(A) binding protein C1 is essential for efficient L1 retrotransposition and affects L1 RNP formation.

Poly(A) binding proteins (PABPs) specifically bind the polyadenosine tail of mRNA and have been shown to be important for RNA polyadenylation, translation initiation, and mRNA stability. Using a modified L1 retrotransposition vector, we examined the effects of two PABPs (encoded by PABPN1 and PABPC1) on the retrotransposition activity of the L1 non-long-terminal-repeat (non-LTR) retrotransposon in both HeLa and HEK293T cells. We demonstrated that knockdown of these two genes by RNA interference (RNAi) effectively reduced L1 retrotransposition by 70 to 80% without significantly changing L1 transcription or translation or the status of the poly(A) tail.

A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver cancer.

The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis.

Characterization of a synthetic human LINE-1 retrotransposon ORFeus-Hs.

Long interspersed elements, type 1(LINE-1, L1) are the most abundant and only active autonomous retrotransposons in the human genome. Native L1 elements are inefficiently expressed because of a transcription elongation defect thought to be caused by high adenosine content in L1 sequences. Previously, we constructed a highly active synthetic mouse L1 element (ORFeus-Mm), partially by reducing the nucleotide composition bias.

Mobilizing diversity: transposable element insertions in genetic variation and disease.

Transposable elements (TEs) comprise a large fraction of mammalian genomes. A number of these elements are actively jumping in our genomes today. As a consequence, these insertions provide a source of genetic variation and, in rare cases, these events cause mutations that lead to disease.

Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model.

Therapeutic strategies based on modulation of microRNA (miRNA) activity hold great promise due to the ability of these small RNAs to potently influence cellular behavior. In this study, we investigated the efficacy of a miRNA replacement therapy for liver cancer. We demonstrate that hepatocellular carcinoma (HCC) cells exhibit reduced expression of miR-26a, a miRNA that is normally expressed at high levels in diverse tissues.

Plug and play modular strategies for synthetic retrotransposons.

Recent progress in L1 biology highlights its role as a major driving force in the evolution of mammalian genome structure and function. This coincides with direct confirmation of the preponderance of long interspersed elements in mammalian genomes at the nucleotide level by large scale sequencing efforts. Two assay systems have been prominently featured in L1 studies over the past decade, which are used to assess L1 activities in cultured cells and transgenic mice respectively.