DICER1 RNase IIIb domain mutations trigger widespread miRNA dysregulation and MAPK activation in pediatric thyroid cancer.

DICER1 is a highly conserved RNase III endoribonuclease essential for the biogenesis of single-stranded mature microRNAs (miRNAs) from stem-loop precursor miRNAs. Somatic mutations in the RNase IIIb domain of DICER1 impair its ability to generate mature 5p miRNAs and are believed to drive tumorigenesis in DICER1 syndrome-associated and sporadic thyroid tumors. However, the DICER1-driven specific changes in miRNAs and resulting changes in gene expression are poorly understood in thyroid tissue. In this study, we profiled the miRNA (n=2,083) and mRNA (n=2,559) transcriptomes of 20 non-neoplastic, 8 adenomatous and 60 pediatric thyroid cancers (13 follicular thyroid cancers [FTC] and 47 papillary thyroid cancers [PTC]) of which 8 had DICER1 RNase IIIb mutations. All DICER1-mutant differentiated thyroid cancers (DTC) were follicular patterned (six follicular variant PTC and two FTC), none had lymph node metastasis. We demonstrate that DICER1 pathogenic somatic mutations were associated with a global reduction of 5p-derived miRNAs, including those particularly abundant in the non-neoplastic thyroid tissue such as let-7 and mir-30 families, known for their tumor suppressor function. There was also an unexpected increase of 3p miRNAs, possibly associated with DICER1 mRNA expression increase in tumors harboring RNase IIIb mutations. These abnormally expressed 3p miRNAs, which are otherwise low or absent in DICER1-wt DTC and non-neoplastic thyroid tissues, make up exceptional markers for malignant thyroid tumors harboring DICER1 RNase IIIb mutations. The extensive disarray in the miRNA transcriptome results in gene expression changes, which were indicative of positive regulation of cell-cycle. Moreover, differentially expressed genes point to increased MAPK signaling output and loss of thyroid differentiation comparable to the RAS-like subgroup of PTC (as coined by The Cancer Genome Atlas), which is reflective of the more indolent clinical behavior of these tumors.

Development of Novel Murine BRAFV600E-Driven Papillary Thyroid Cancer Cell Lines for Modeling of Disease Progression and Preclinical Evaluation of Therapeutics.

The Cancer Genome Atlas study in thyroid cancer exposed the genomic landscape of ~500 PTCs and revealed BRAFV600E-mutant tumors as having different prognosis, contrasting indolent cases and those with more invasive disease. Here, we describe the generation and characterization of six novel BRAFV600E-driven papillary thyroid cancer (PTC) cell lines established from a BrafV600E+/-/Pten+/-/TPO-Cre mouse model that spontaneously develop thyroid tumors. The novel cell lines were obtained from animals representing a range of developmental stages and both sexes, with the goal of establishing a heterogeneous panel of PTC cell lines sharing a common driver mutation. These cell lines recapitulate the genetics and diverse histopathological features of BRAFV600E-driven PTC, exhibiting differing degrees of growth, differentiation, and invasive potential that may help define mechanisms of pathogenesis underlying the heterogeneity present in the patient population. We demonstrate that these cell lines can be used for a variety of in vitro applications and can maintain the potential for in vivo transplantation into immunocompetent hosts. We believe that these novel cell lines will provide powerful tools for investigating the molecular basis of thyroid cancer progression and will lead to the development of more personalized diagnostic and treatment strategies for BRAFV600E-driven PTC.

Hormonal Crosstalk Between Thyroid and Breast Cancer.

Differentiated thyroid cancer and breast cancer account for a significant portion of endocrine-related malignancies and predominately affect women. As hormonally responsive tissues, the breast and thyroid share endocrine signaling. Breast cells are responsive to thyroid hormone signaling and are affected by altered thyroid hormone levels. Thyroid cells are responsive to sex hormones, particularly estrogen, and undergo protumorigenic processes upon estrogen stimulation. Thyroid and sex hormones also display significant transcriptional crosstalk that influences oncogenesis and treatment sensitivity. Obesity-related adipocyte alterations-adipocyte estrogen production, inflammation, feeding hormone dysregulation, and metabolic syndromes-promote hormonal alterations in breast and thyroid tissues. Environmental toxicants disrupt endocrine systems, including breast and thyroid homeostasis, and influence pathologic processes in both organs through hormone mimetic action. In this brief review, we discuss the hormonal connections between the breast and thyroid and perspectives on hormonal therapies for breast and thyroid cancer. Future research efforts should acknowledge and further explore the hormonal crosstalk of these tissues in an effort to further understand the prevalence of thyroid and breast cancer in women and to identify potential therapeutic options.

NTRK-fusions in pediatric thyroid tumors: Current state and future perspectives.

Pediatric and adult papillary thyroid cancer (PTC) share many similar oncogenic drivers, but differ in the pathological features and outcomes of the disease. The most frequent genetic alterations in adult PTCs are mutually exclusive point mutations in BRAF or the RAS family. In pediatric PTC, fusion oncogenes involving chromosomal translocations in tyrosine kinase (TK) receptors, most commonly RET and NTRK, are the most common genetic alterations observed. This review of the literature describes the current state of translational research in pediatric NTRK-driven thyroid cancer and highlights opportunities to improve our understanding and current models of pediatric PTC.

The clinical aspect of NTRK-fusions in pediatric papillary thyroid cancer.

Although adult and pediatric papillary thyroid cancer (PTC) share similar oncogenic drivers, they differ in the pathological features and outcomes of the disease. In adults with PTC, the most frequent genetic alterations are mutually exclusive point mutations in BRAFV600E or the RAS family with BRAFV600E commonly associated with invasive disease and decreased response to radioiodine therapy. In pediatric PTC, fusion oncogenes involving chromosomal translocations in tyrosine kinase (TK) receptors, most commonly RET and NTRK, are often found in patients with lateral neck and distant metastases. This brief report reviews clinical data from a single-institute's cohort of NTRK-driven pediatric PTC cases with an updated review of the literature and comparison to adult NTRK-driven PTC.

CAR-T cell-mediated depletion of immunosuppressive tumor-associated macrophages promotes endogenous antitumor immunity and augments adoptive immunotherapy.

The immunosuppressive tumor microenvironment (TME) represents a major barrier for effective immunotherapy. Tumor-associated macrophages (TAMs) are highly heterogeneous and plastic cell components of the TME which can either promote tumor progression (M2-like) or boost antitumor immunity (M1-like). Here, we demonstrate that a subset of TAMs that express folate receptor ß (FRß) possess an immunosuppressive M2-like profile. In syngeneic tumor mouse models, chimeric antigen receptor (CAR)-T cell-mediated selective elimination of FRß+ TAMs in the TME results in an enrichment of pro-inflammatory monocytes, an influx of endogenous tumor-specific CD8+ T cells, delayed tumor progression, and prolonged survival. Preconditioning of the TME with FRß-specific CAR-T cells also improves the effectiveness of tumor-directed anti-mesothelin CAR-T cells, while simultaneous co-administration of both CAR products does not. These results highlight the pro-tumor role of FRß+ TAMs in the TME and the therapeutic implications of TAM-depleting agents as preparative adjuncts to conventional immunotherapies that directly target tumor antigens.

PKCε regulates Rho GTPases and actin cytoskeleton reorganization in non-small cell lung cancer cells.

Oncogenic protein kinase C epsilon (PKCε) promotes the formation of membrane ruffles and motility in non-small cell lung cancer (NSCLC) cells. We found that PKCε is down-regulated when NSCLC cells undergo epithelial-to-mesenchymal transition (EMT) in response to TGF-ß, thus becoming dispensable for migration and invasion in the mesenchymal state. PKCε silencing or inhibition leads to stress fibre formation, suggesting that this kinase negatively regulates RhoA activity. Ruffle formation induced by PKCε activation in the epithelial state is dependent on PI3K, but does not involve the PI3K-dependent Rac-GEFs Ect2, Trio, Vav2 or Tiam1, suggesting alternative Rac-GEFs as mediators of this response. In the proposed model, PKCε acts as a rheostat for Rho GTPases that differs in the epithelial and mesenchymal states.

Single residue in CD28-costimulated CAR-T cells limits long-term persistence and antitumor durability.

Chimeric antigen receptor-T (CAR-T) cell therapies can eliminate relapsed and refractory tumors, but the durability of antitumor activity requires in vivo persistence. Differential signaling through the CAR costimulatory domain can alter the T cell metabolism, memory differentiation, and influence long-term persistence. CAR-T cells costimulated with 4-1BB or ICOS persist in xenograft models but those constructed with CD28 exhibit rapid clearance. Here, we show that a single amino acid residue in CD28 drove T cell exhaustion and hindered the persistence of CD28-based CAR-T cells and changing this asparagine to phenylalanine (CD28-YMFM) promoted durable antitumor control. In addition, CD28-YMFM CAR-T cells exhibited reduced T cell differentiation and exhaustion as well as increased skewing toward Th17 cells. Reciprocal modification of ICOS-containing CAR-T cells abolished in vivo persistence and antitumor activity. This finding suggests modifications to the costimulatory domains of CAR-T cells can enable longer persistence and thereby improve antitumor response.

Identification of a truncated ß1-chimaerin variant that inactivates nuclear Rac1.

ß1-chimaerin belongs to the chimaerin family of GTPase-activating proteins (GAPs) and is encoded by the CHN2 gene, which also encodes the ß2- and ß3-chimaerin isoforms. All chimaerin isoforms have a C1 domain that binds diacylglycerol as well as tumor-promoting phorbol esters and a catalytic GAP domain that inactivates the small GTPase Rac. Nuclear Rac has emerged as a key regulator of various cell functions, including cell division, and has a pathological role by promoting tumorigenesis and metastasis. However, how nuclear Rac is regulated has not been fully addressed. Here, using several approaches, including siRNA-mediated gene silencing, confocal microscopy, and subcellular fractionation, we identified a nuclear variant of ß1-chimaerin, ß1-Δ7p-chimaerin, that participates in the regulation of nuclear Rac1. We show that ß1-Δ7p-chimaerin is a truncated variant generated by alternative splicing at a cryptic splice site in exon 7. We found that, unlike other chimaerin isoforms, ß1-Δ7p-chimaerin lacks a functional C1 domain and is not regulated by diacylglycerol. We found that ß1-Δ7p-chimaerin localizes to the nucleus via a nuclear localization signal in its N terminus. We also identified a key nuclear export signal in ß1-chimaerin that is absent in ß1-Δ7p-chimaerin, causing nuclear retention of this truncated variant. Functionally analyses revealed that ß1-Δ7p-chimaerin inactivates nuclear Rac and negatively regulates the cell cycle. Our results provide important insights into the diversity of chimaerin Rac-GAP regulation and function and highlight a potential mechanism of nuclear Rac inactivation that may play significant roles in pathologies such as cancer.

Differential Regulation of Gene Expression in Lung Cancer Cells by Diacyglycerol-Lactones and a Phorbol Ester Via Selective Activation of Protein Kinase C Isozymes.

Despite our extensive knowledge on the biology of protein kinase C (PKC) and its involvement in disease, limited success has been attained in the generation of PKC isozyme-specific modulators acting via the C1 domain, the binding site for the lipid second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. Synthetic efforts had recently led to the identification of AJH-836, a DAG-lactone with preferential affinity for novel isozymes (nPKCs) relative to classical PKCs (cPKCs). Here, we compared the ability of AJH-836 and a prototypical phorbol ester (phorbol 12-myristate 13-acetate, PMA) to induce changes in gene expression in a lung cancer model. Gene profiling analysis using RNA-Seq revealed that PMA caused major changes in gene expression, whereas AJH-836 only induced a small subset of genes, thus providing a strong indication for a major involvement of cPKCs in their control of gene expression. MMP1, MMP9, and MMP10 were among the genes most prominently induced by PMA, an effect impaired by RNAi silencing of PKCα, but not PKCδ or PKCε. Comprehensive gene signature analysis and bioinformatics efforts, including functional enrichment and transcription factor binding site analyses of dysregulated genes, identified major differences in pathway activation and transcriptional networks between PMA and DAG-lactones. In addition to providing solid evidence for the differential involvement of individual PKC isozymes in the control of gene expression, our studies emphasize the importance of generating targeted C1 domain ligands capable of differentially regulating PKC isozyme-specific function in cellular models.

Distinctive requirement of PKCε in the control of Rho GTPases in epithelial and mesenchymally transformed lung cancer cells.

Diacylglycerol (DAG)/phorbol ester-regulated protein kinase C (PKC) isozymes have been widely linked to tumor promotion and the development of a metastatic phenotype. PKCε, an oncogenic member of the PKC family, is abnormally overexpressed in lung cancer and other cancer types. This kinase plays significant roles in proliferation, survival, and migration; however, its role in epithelial-to-mesenchymal transition (EMT) has been scarcely studied. Silencing experiments in non-small lung cancer (NSCLC) cells revealed that PKCε or other DAG-regulated PKCs (PKCα and PKCδ) were dispensable for the acquisition of a mesenchymal phenotype induced by transforming growth factor beta (TGF-ß). Unexpectedly, we found a nearly complete down-regulation of PKCε expression in TGF-ß-mesenchymally transformed NSCLC cells. PMA and AJH-836 (a DAG-mimetic that preferentially activates PKCε) promote ruffle formation in NSCLC cells via Rac1, however they fail to induce these morphological changes in TGF-ß-mesenchymally transformed cells despite their elevated Rac1 activity. Several Rac guanine nucleotide exchange-factors (Rac-GEFs) were also up-regulated in TGF-ß-treated NSCLC cells, including Trio and Tiam2, which were required for cell motility. Lastly, we found that silencing or inhibiting PKCε enhances RhoA activity and stress fiber formation, a phenotype also observed in TGF-ß-transformed cells. Our studies established a distinctive involvement of PKCε in epithelial and mesenchymal NSCLC cells, and identified a complex interplay between PKCε and small GTPases that contributes to regulation of NSCLC cell morphology and motile activity.

The role of Rac in tumor susceptibility and disease progression: from biochemistry to the clinic.

The family of Rho GTPases are involved in the dynamic control of cytoskeleton reorganization and other fundamental cellular functions, including growth, motility, and survival. Rac1, one of the best characterized Rho GTPases, is an established effector of receptors and an important node in signaling networks crucial for tumorigenesis and metastasis. Rac1 hyperactivation is common in human cancer and could be the consequence of overexpression, abnormal upstream inputs, deregulated degradation, and/or anomalous intracellular localization. More recently, cancer-associated gain-of-function mutations in Rac1 have been identified which contribute to tumor phenotypes and confer resistance to targeted therapies. Deregulated expression/activity of Rac guanine nucleotide exchange factors responsible for Rac activation has been largely associated with a metastatic phenotype and drug resistance. Translating our extensive knowledge in Rac pathway biochemistry into a clinical setting still remains a major challenge; nonetheless, remarkable opportunities for cancer therapeutics arise from promising lead compounds targeting Rac and its effectors.

P-Rex1 is dispensable for Erk activation and mitogenesis in breast cancer.

Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 1 (P-Rex1) is a key mediator of growth factor-induced activation of Rac1, a small GTP-binding protein widely implicated in actin cytoskeleton reorganization. This Guanine nucleotide Exchange Factor (GEF) is overexpressed in human luminal breast cancer, and its expression associates with disease progression, metastatic dissemination and poor outcome. Despite the established contribution of P-Rex1 to Rac activation and cell locomotion, whether this Rac-GEF has any relevant role in mitogenesis has been a subject of controversy. To tackle the discrepancies among various reports, we carried out an exhaustive analysis of the potential involvement of P-Rex1 on the activation of the mitogenic Erk pathway. Using a range of luminal breast cancer cellular models, we unequivocally showed that silencing P-Rex1 (transiently, stably, using multiple siRNA sequences) had no effect on the phospho-Erk response upon stimulation with growth factors (EGF, heregulin, IGF-I) or a GPCR ligand (SDF-1). The lack of involvement of P-Rex1 in Erk activation was confirmed at the single cell level using a fluorescent biosensor of Erk kinase activity. Depletion of P-Rex1 from breast cancer cells failed to affect cell cycle progression, cyclin D1 induction, Akt activation and apoptotic responses. In addition, mammary-specific P-Rex1 transgenic mice (MMTV-P-Rex1) did not show any obvious hyperproliferative phenotype. Therefore, despite its crucial role in Rac1 activation and cell motility, P-Rex1 is dispensable for mitogenic or survival responses in breast cancer cells.

Characterization of AJH-836, a diacylglycerol-lactone with selectivity for novel PKC isozymes.

Diacylglycerol (DAG) is a key lipid second messenger downstream of cellular receptors that binds to the C1 domain in many regulatory proteins. Protein kinase C (PKC) isoforms constitute the most prominent family of signaling proteins with DAG-responsive C1 domains, but six other families of proteins, including the chimaerins, Ras-guanyl nucleotide-releasing proteins (RasGRPs), and Munc13 isoforms, also play important roles. Their significant involvement in cancer, immunology, and neurobiology has driven intense interest in the C1 domain as a therapeutic target. As with other classes of targets, however, a key issue is the establishment of selectivity. Here, using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) competition binding assays, we found that a synthetic DAG-lactone, AJH-836, preferentially binds to the novel PKC isoforms PKCδ and PKCϵ relative to classical PKCα and PKCßII. Assessment of intracellular translocation, a hallmark for PKC activation, revealed that AJH-836 treatment stimulated a striking preferential redistribution of PKCϵ to the plasma membrane relative to PKCα. Moreover, unlike with the prototypical phorbol ester phorbol 12-myristate 13-acetate (PMA), prolonged exposure of cells to AJH-836 selectively down-regulated PKCδ and PKCϵ without affecting PKCα expression levels. Biologically, AJH-836 induced major changes in cytoskeletal reorganization in lung cancer cells, as determined by the formation of membrane ruffles, via activation of novel PKCs. We conclude that AJH-836 represents a C1 domain ligand with PKC-activating properties distinct from those of natural DAGs and phorbol esters. Our study supports the feasibility of generating selective C1 domain ligands that promote novel biological response patterns.

Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation.

Successful tumor eradication by chimeric antigen receptor-expressing (CAR-expressing) T lymphocytes depends on CAR T cell persistence and effector function. We hypothesized that CD4+ and CD8+ T cells may exhibit distinct persistence and effector phenotypes, depending on the identity of specific intracellular signaling domains (ICDs) used to generate the CAR. First, we demonstrate that the ICOS ICD dramatically enhanced the in vivo persistence of CAR-expressing CD4+ T cells that, in turn, increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. These data indicate that persistence of CD8+ T cells was highly dependent on a helper effect provided by the ICD used to redirect CD4+ T cells. Second, we discovered that combining ICOS and 4-1BB ICDs in a third-generation CAR displayed superior antitumor effects and increased persistence in vivo. Interestingly, we found that the membrane-proximal ICD displayed a dominant effect over the distal domain in third-generation CARs. The optimal antitumor and persistence benefits observed in third-generation ICOSBBz CAR T cells required the ICOS ICD to be positioned proximal to the cell membrane and linked to the ICOS transmembrane domain. Thus, CARs with ICOS and 4-1BB ICD demonstrate increased efficacy in solid tumor models over our current 4-1BB-based CAR and are promising therapeutics for clinical testing.

The P-Rex1/Rac signaling pathway as a point of convergence for HER/ErbB receptor and GPCR responses.

Guanine nucleotide Exchange Factors (GEFs) are responsible for mediating GDP/GTP exchange for specific small G proteins, such as Rac. There has been substantial evidence for the involvement of Rac-GEFs in the control of cancer cell migration and metastatic progression. We have previously established that the Rac-GEF P-Rex1 is a mediator of actin cytoskeleton rearrangements and cell motility in breast cancer cells downstream of HER/ErbB receptors and the G-Protein Coupled Receptor (GPCR) CXCR4. P-Rex1 is highly expressed in luminal A and B breast cancer compared to normal mammary tissue, whereas expression is very low in basal breast cancer, and its expression correlates with the appearance of metastasis in patients. Here, we discuss the involvement of P-Rex1 as an effector of oncogenic/metastatic receptors in breast cancer and underscore its relevance in the convergence of receptor-triggered motile signals. In addition, we provide an overview of our recent findings describing a cross-talk between HER/ErbB receptors and CXCR4, and how this impacts on the activation of P-Rex1/Rac1 signaling, as well as highlight challenges that lie ahead. We propose a model in which P-Rex1 acts as a crucial node for the integration of upstream inputs from HER/ErbB receptors and CXCR4 in luminal breast cancer cells.

Protein kinase C in cancer: The top five unanswered questions.

Few kinases have been studied as extensively as protein kinase C (PKC), particularly in the context of cancer. As major cellular targets for the phorbol ester tumor promoters and diacylglycerol (DAG), a second messenger generated by stimulation of membrane receptors, PKC isozymes play major roles in the control of signaling pathways associated with proliferation, migration, invasion, tumorigenesis, and metastasis. However, despite decades of research, fundamental questions remain to be answered or are the subject of intense controversy. Primary among these unresolved issues are the role of PKC isozymes as either tumor promoter or tumor suppressor kinases and the incomplete understanding on isozyme-specific substrates and effectors. The involvement of PKC isozymes in cancer progression needs to be reassessed in the context of specific oncogenic and tumor suppressing alterations. In addition, there are still major hurdles in addressing isozyme-specific function due to the limited specificity of most pharmacological PKC modulators and the lack of validated predictive biomarkers for response, which impacts the translation of these agents to the clinic. In this review we focus on key controversial issues and upcoming challenges, with the expectation that understanding the intricacies of PKC function will help fulfill the yet unsuccessful promise of targeting PKCs for cancer therapeutics.

Heregulin/ErbB3 Signaling Enhances CXCR4-Driven Rac1 Activation and Breast Cancer Cell Motility via Hypoxia-Inducible Factor 1α.

The growth factor heregulin (HRG), a ligand of ErbB3 and ErbB4 receptors, contributes to breast cancer development and the promotion of metastatic disease, and its expression in breast tumors has been associated with poor clinical outcome and resistance to therapy. In this study, we found that breast cancer cells exposed to sustained HRG treatment show markedly enhanced Rac1 activation and migratory activity in response to the CXCR4 ligand SDF-1/CXCL12, effects mediated by P-Rex1, a Rac-guanine nucleotide exchange factor (GEF) aberrantly expressed in breast cancer. Notably, HRG treatment upregulates surface expression levels of CXCR4, a G protein-coupled receptor (GPCR) implicated in breast cancer metastasis and an indicator of poor prognosis in breast cancer patients. A detailed mechanistic analysis revealed that CXCR4 upregulation and sensitization of the Rac response/motility by HRG are mediated by the transcription factor hypoxia-inducible factor 1α (HIF-1α) via ErbB3 and independently of ErbB4. HRG caused prominent induction in the nuclear expression of HIF-1α, which transcriptionally activates the CXCR4 gene via binding to a responsive element located in positions -1376 to -1372 in the CXCR4 promoter, as revealed by mutagenesis analysis and chromatin immunoprecipitation (ChIP). Our results uncovered a novel function for ErbB3 in enhancing breast cancer cell motility and sensitization of the P-Rex1/Rac1 pathway through HIF-1α-mediated transcriptional induction of CXCR4.

A new role of the Rac-GAP ß2-chimaerin in cell adhesion reveals opposite functions in breast cancer initiation and tumor progression.

ß2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer but its precise function in mammary tumorigenesis in vivo is unknown. Here, we study for the first time the role of ß2-chimaerin in breast cancer using a mouse model and describe an unforeseen role for this protein in epithelial cell-cell adhesion. We demonstrate that expression of ß2-chimaerin in breast cancer epithelial cells reduces E-cadherin protein levels, thus loosening cell-cell contacts. In vivo, genetic ablation of ß2-chimaerin in the MMTV-Neu/ErbB2 mice accelerates tumor onset, but delays tumor progression. Finally, analysis of clinical databases revealed an inverse correlation between ß2-chimaerin and E-cadherin gene expressions in Her2+ breast tumors. Furthermore, breast cancer patients with low ß2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times. Overall, our data redefine the role of ß2-chimaerin as tumor suppressor and provide the first in vivo evidence of a dual function in breast cancer, suppressing tumor initiation but favoring tumor progression.