Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

Cell death-induced immunogenicity enhances chemoimmunotherapeutic response by converting immune-excluded into T-cell inflamed bladder tumors.

Chemoimmunotherapy has recently failed to demonstrate significant clinical benefit in advanced bladder cancer patients; and the mechanism(s) underlying such suboptimal response remain elusive. To date, most studies have focused on tumor-intrinsic properties that render them "immune-excluded". Here, we explore an alternative, drug-induced mechanism that impedes therapeutic response via disrupting the onset of immunogenic cell death. Using two immune-excluded syngeneic mouse models of muscle-invasive bladder cancer (MIBC), we show that platinum-based chemotherapy diminishes CD8+ T cell tumor infiltration and constraines their antitumoral activity, despite expression of activation markers IFNγ and granzyme B. Mechanistically, chemotherapy induces the release of prostaglandin E2 (PGE2) from dying cancer cells, which is an inhibitory damage-associated molecular pattern (iDAMP) that hinderes dendritic cell maturation. Upon pharmaceutical blockade of PGE2 release, CD8+ T cells become tumoricidal and display an intraepithelial-infiltrating (or inflamed) pattern. This "iDAMP blockade" approach synergizes with chemotherapy and sensitizes bladder tumors towards anti-PD1 immune checkpoint inhibitor therapy. These findings provide a compelling rationale to evaluate this drug combination in future clinical trials.

Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression.

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.

Inhibitory DAMPs in immunogenic cell death and its clinical implications.

Dying (or dead) cells are increasingly recognized to impose significant biological influence within their tissues of residence-exerting paracrine effects through proteins and metabolites that are expressed or secreted during cellular demise. For example, certain molecules function as potent mitogens, promoting the repopulation of neighboring epithelial cells. And other myriad of factors-classified as damage-associated molecular patterns (DAMPs)-function as "find me" (attractant), "eat me" (engulfment), or "danger" (activation) signals for recruiting and activating effector immune cells (e.g., dendritic cells) to initiate inflammation. Since the discovery of immunogenic cell death (ICD), the current dogma posits DAMPs as immunological adjuvants for innate immune cell mobilization and activation, which ultimately leads to the antitumoral cross-priming of CD8+ T cells. However, what is currently unknown is how these immunostimulatory DAMPs are counteracted to avoid immune-overactivation. Our recent work builds on these fundamentals and introduces prostaglandin E2 (PGE2) as an 'inhibitory' DAMP-a new variable to the ICD equation. Prostaglandin E2 functions as an immunosuppressive counterpoise of adjuvant DAMPs; and thus, mechanistically precludes ICD. Furthermore, the long-debated immunogenicity of gemcitabine chemotherapy was revealed to be contingent on inhibitory DAMP blockade and not due to its inability to promote DAMP expression (i.e., calreticulin) as previously reported. These findings were intriguing. First, despite the presence of gemcitabine-induced hallmark DAMPs, the inhibitory DAMP (i.e., PGE2) was sufficient to hinder the ICD-induced antitumoral immune response (Fig. 1a). And second, rather than pharmacologically substantiating immunostimulatory DAMPs as conventionally approached, the mitigation of the inhibitory DAMP-tipping the immunostimulatory and inhibitory DAMP balance in favor of immunostimulatory DAMPs-was sufficient to render the cell death immunogenic and converted gemcitabine into an ICD-inducing therapy (Fig. 1b). In this microreview, we extrapolate our findings and implicate the value of inhibitory DAMP(s) in drug discovery, its use for clinical prognosis, and as target(s) for therapeutic intervention.

Estrogen Receptor ß-Mediated Inhibition of Actin-Based Cell Migration Suppresses Metastasis of Inflammatory Breast Cancer.

Inflammatory breast cancer (IBC) is a highly metastatic breast carcinoma with high frequency of estrogen receptor α (ERα) negativity. Here we explored the role of the second ER subtype, ERß, and report expression in IBC tumors and its correlation with reduced metastasis. Ablation of ERß in IBC cells promoted cell migration and activated gene networks that control actin reorganization, including G-protein-coupled receptors and downstream effectors that activate Rho GTPases. Analysis of preclinical mouse models of IBC revealed decreased metastasis of IBC tumors when ERß was expressed or activated by chemical agonists. Our findings support a tumor-suppressive role of ERß by demonstrating the ability of the receptor to inhibit dissemination of IBC cells and prevent metastasis. On the basis of these findings, we propose ERß as a potentially novel biomarker and therapeutic target that can inhibit IBC metastasis and reduce its associated mortality. SIGNIFICANCE: These findings demonstrate the capacity of ERß to elicit antimetastatic effects in highly aggressive inflammatory breast cancer and propose ERß and the identified associated genes as potential therapeutic targets in this disease.

Collagen-rich airway smooth muscle cells are a metastatic niche for tumor colonization in the lung.

Metastases account for the majority of cancer deaths. While certain steps of the metastatic cascade are well characterized, identification of targets to block this process remains a challenge. Host factors determining metastatic colonization to secondary organs are particularly important for exploration, as those might be shared among different cancer types. Here, we showed that bladder tumor cells expressing the collagen receptor, CD167a, responded to collagen I stimulation at the primary tumor to promote local invasion and utilized the same receptor to preferentially colonize at airway smooth muscle cells (ASMCs)-a rich source of collagen III in lung. Morphologically, COL3-CD167a-driven metastatic foci are uniquely distinct from typical lung alveolar metastatic lesions and exhibited activation of the CD167a-HSP90-Stat3 axis. Importantly, metastatic lung colonization could be abrogated using an investigational drug that attenuates Stat3 activity, implicating this seed-and-soil interaction as a therapeutic target for eliminating lung metastasis.

ERß alters the chemosensitivity of luminal breast cancer cells by regulating p53 function.

Estrogen receptor α (ERα)-positive breast cancers tend to develop resistance to both endocrine therapy and chemotherapy. Despite recent progress in defining molecular pathways that confer endocrine resistance, the mechanisms that regulate chemotherapy response in luminal tumors remain largely elusive. Luminal tumors often express wild-type p53 that is a major determinant of the cellular DNA damage response. Similar to p53, the second ER subtype, ERß, has been reported to inhibit breast tumorigenesis by acting alone or in collaboration with p53. However, a synergistic mechanism of action has not been described. Here, we suggest that ERß relies on p53 to elicit its tumor repressive actions in ERα-positive breast cancer cells. Upregulation of ERß and treatment with ERß agonists potentiates the tumor suppressor function of p53 resulting in decreased survival. This effect requires molecular interaction between the two proteins that disrupts the inhibitory action of ERα on p53 leading to increased transcriptional activity of p53. In addition, we show that the same interaction alters the chemosensitivity of endocrine-resistant cells including their response to tamoxifen therapy. Our results suggest a collaboration of ERß and p53 tumor suppressor activity in breast cancer cells that indicates the importance of ligand-regulated ERß as a tool to target p53 activity and improve the clinical management of resistant disease.

GLUT12 promotes prostate cancer cell growth and is regulated by androgens and CaMKK2 signaling.

Despite altered metabolism being an accepted hallmark of cancer, it is still not completely understood which signaling pathways regulate these processes. Given the central role of androgen receptor (AR) signaling in prostate cancer, we hypothesized that AR could promote prostate cancer cell growth in part through increasing glucose uptake via the expression of distinct glucose transporters. Here, we determined that AR directly increased the expression of SLC2A12, the gene that encodes the glucose transporter GLUT12. In support of these findings, gene signatures of AR activity correlated with SLC2A12 expression in multiple clinical cohorts. Functionally, GLUT12 was required for maximal androgen-mediated glucose uptake and cell growth in LNCaP and VCaP cells. Knockdown of GLUT12 also decreased the growth of C4-2, 22Rv1 and AR-negative PC-3 cells. This latter observation corresponded with a significant reduction in glucose uptake, indicating that additional signaling mechanisms could augment GLUT12 function in an AR-independent manner. Interestingly, GLUT12 trafficking to the plasma membrane was modulated by calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-5'-AMP-activated protein kinase (AMPK) signaling, a pathway we previously demonstrated to be a downstream effector of AR. Inhibition of CaMKK2-AMPK signaling decreased GLUT12 translocation to the plasma membrane by inhibiting the phosphorylation of TBC1D4, a known regulator of glucose transport. Further, AR increased TBC1D4 expression. Correspondingly, expression of TBC1D4 correlated with AR activity in prostate cancer patient samples. Taken together, these data demonstrate that prostate cancer cells can increase the functional levels of GLUT12 through multiple mechanisms to promote glucose uptake and subsequent cell growth.

ICG-001 Exerts Potent Anticancer Activity Against Uveal Melanoma Cells.

Purpose: Uveal melanoma (UM) is uniformly refractory to all available systemic chemotherapies, thus creating an urgent need for novel therapeutics. In this study, we investigated the sensitivity of UM cells to ICG-001, a small molecule reported to suppress the Wnt/ß-catenin-mediated transcriptional program. Methods: We used a panel of UM cell lines to examine the effects of ICG-001 on cellular proliferation, migration, and gene expression. In vivo efficacy of ICG-001 was evaluated in a UM xenograft model. Results: ICG-001 exerted strong antiproliferative activity against UM cells, leading to cell cycle arrest, apoptosis, and inhibition of migration. Global gene expression profiling revealed strong suppression of genes associated with cell cycle proliferation, DNA replication, and G1/S transition. Gene set enrichment analysis revealed that ICG-001 suppressed Wnt, mTOR, and MAPK signaling. Strikingly, ICG-001 suppressed the expression of genes associated with UM aggressiveness, including CDH1, CITED1, EMP1, EMP3, SDCBP, and SPARC. Notably, the transcriptomic footprint of ICG-001, when applied to a UM patient dataset, was associated with better clinical outcome. Lastly, ICG-001 exerted anticancer activity against a UM tumor xenograft in mice. Conclusions: Using in vitro and in vivo experiments, we demonstrate that ICG-001 has strong anticancer activity against UM cells and suppresses transcriptional programs critical for the cancer cell. Our results suggest that ICG-001 holds promise and should be examined further as a novel therapeutic agent for UM.

Prognostic Power of a Tumor Differentiation Gene Signature for Bladder Urothelial Carcinomas.

Background: Muscle-invasive bladder cancers (MIBCs) cause approximately 150 000 deaths per year worldwide. Survival for MIBC patients is heterogeneous, with no clinically validated molecular markers that predict clinical outcome. Non-MIBCs (NMIBCs) generally have favorable outcome; however, a portion progress to MIBC. Hence, development of a prognostic tool that can guide decision-making is crucial for improving clinical management of bladder urothelial carcinomas. Methods: Tumor grade is defined by pathologic evaluation of tumor cell differentiation, and it often associates with clinical outcome. The current study extrapolates this conventional wisdom and combines it with molecular profiling. We developed an 18-gene signature that molecularly defines urothelial cellular differentiation, thus classifying MIBCs and NMIBCs into two subgroups: basal and differentiated. We evaluated the prognostic capability of this "tumor differentiation signature" and three other existing gene signatures including the The Cancer Genome Atlas (TCGA; 2707 genes), MD Anderson Cancer Center (MDA; 2252 genes/2697 probes), and University of North Carolina at Chapel Hill (UNC; 47 genes) using five gene expression data sets derived from MIBC and NMIBC patients. All statistical tests were two-sided. Results: The tumor differentiation signature demonstrated consistency and statistical robustness toward stratifying MIBC patients into different overall survival outcomes (TCGA cohort 1, P = .03; MDA discovery, P = .009; MDA validation, P = .01), while the other signatures were not as consistent. In addition, we analyzed the progression (Ta/T1 progressing to ≥T2) probability of NMIBCs. NMIBC patients with a basal tumor differentiation signature associated with worse progression outcome (P = .008). Gene functional term enrichment and gene set enrichment analyses revealed that genes involved in the biologic process of immune response and inflammatory response are among the most elevated within basal bladder cancers, implicating them as candidates for immune checkpoint therapies. Conclusions: These results provide definitive evidence that a biology-prioritizing clustering methodology generates meaningful insights into patient stratification and reveals targetable molecular pathways to impact future therapeutic approach.

ERß Sensitizes NSCLC to Chemotherapy by Regulating DNA Damage Response.

The expression of wild-type estrogen receptor ß (ESR2/ERß1) correlates with clinical outcome in patients with non-small cell lung cancer (NSCLC). However, the molecular mechanism that accounts for this association is currently poorly understood. ERß1 was previously linked to chemotherapy response in patients with breast cancer and in breast cancer cells. The effect of the receptor in NSCLC cells after chemotherapy treatment, a common remedy for advanced NSCLC, has not been studied. Here, upregulation of ERß1 increases the sensitivity of NSCLC cells to treatment with doxorubicin and etoposide. This effect was primarily observed in p53-defecient NSCLC cells. In these cells, ERß1 either enhanced G2-M cell-cycle arrest by activating the checkpoint kinase 1 (Chk1) and altering downstream signaling or induced apoptosis. The expression of p63 target genes that control G2-M checkpoint activation was altered by ERß1 suggesting an ERß1-p63 transcriptional cooperation in lung cancer cells that affects DNA damage response (DDR). These results suggest involvement of ERß1 in the mechanism that regulates DNA damage response in NSCLC cells and support the potential predictive and therapeutic value of the receptor in clinical management of the disease.Implications: This study demonstrating the impact of ERß1 on chemosensitivity of NSCLC cells suggests the predictive value of the receptor for successful response of tumors to chemotherapy and the potential benefit of chemotherapy-treated patients from the use of ER ligands. Mol Cancer Res; 16(2); 233-42. ©2017 AACR.

Somatic loss of estrogen receptor beta and p53 synergize to induce breast tumorigenesis.

BACKGROUND: Upregulation of estrogen receptor beta (ERß) in breast cancer cells is associated with epithelial maintenance, decreased proliferation and invasion, and a reduction in the expression of the receptor has been observed in invasive breast tumors. However, proof of an association between loss of ERß and breast carcinogenesis is still missing. METHODS: To study the role of ERß in breast oncogenesis, we generated mouse conditional mutants with specific inactivation of ERß and p53 in the mammary gland epithelium. For epithelium-specific knockout of ERß and p53, ERß F/F and p53 F/F mice were crossed to transgenic mice that express the Cre recombinase under the control of the human keratin 14 promoter. RESULTS: Somatic loss of ERß significantly accelerated formation of p53-deficient mammary tumors. Loss of the receptor also resulted in the development of less differentiated carcinomas with stronger spindle cell morphology and decreased expression of luminal epithelial markers. CONCLUSIONS: Our results show that synergism between ERß and p53 inactivation functions to determine important aspects of breast oncogenesis and cancer progression.

ERß decreases the invasiveness of triple-negative breast cancer cells by regulating mutant p53 oncogenic function.

Most (80%) of the triple-negative breast cancers (TNBCs) express mutant p53 proteins that acquire oncogenic activities including promoting metastasis. We previously showed that wild-type ERß (ERß1) impedes epithelial to mesenchymal transition (EMT) and decreases the invasiveness of TNBC cells. In the present study we searched for signaling pathways that ERß1 uses to inhibit EMT and invasion in TNBC cells. We show that ERß1 binds to and opposes the transcriptional activity of mutant p53 at the promoters of genes that regulate metastasis. p63 that transcriptionally cooperates with mutant p53 also binds to ERß1. Downregulation of p63 represses the epithelial phenotype of ERß1-expressing cells and alters the expression of mutant p53 target genes. These results describe a novel mechanism through which ERß1 can disturb oncogenic signals to inhibit aggressiveness in TNBCs.

Pleiotropic signaling evoked by tumor necrosis factor in podocytes.

TNF has been implicated in glomerular diseases, but its actions on podocytes are not well understood. Endogenous TNF expression is markedly increased in mouse podocytes exposed to sera from patients with recurrent focal segmental glomerulosclerosis, and TNF is able to increase its own expression in these cells. Exposure of podocytes to TNF increased phosphorylation of NF-κB p65-RelA followed by increased tyrosine phosphorylation of STAT3. STAT3 activation was blocked by the NF-κB inhibitor JSH-23 and by the STAT3 inhibitor stattic, whereas TNF-evoked NF-κB activation was not affected by stattic. TNF treatment increased nuclear accumulation of nuclear factor of activated T cells (NFAT)c1 in podocytes, a process that occurred downstream of STAT3 activation. TNF also increased expression of cyclin D1 but had no effect on cyclin-dependent kinase 4, p27(kip), or podocin. Despite its effects on cyclin D1, TNF treatment for up to 72 h did not cause podocytes to reenter the cell cycle. TNF increased total expression of transient receptor potential (TRP)C6 channels through a pathway dependent on NFATc1 and increased the steady-state expression of TRPC6 subunits on the podocyte cell surface. TNF effects on TRPC6 trafficking required ROS. Consistent with this, La(3+)-sensitive cationic currents activated by a diacylglycerol analog were increased in TNF-treated cells. The effects of TNF on NFATc1 and TRPC6 expression were blocked by cyclosporine A but were not blocked by the pan-TRP inhibitor SKF-96365. TNF therefore influences multiple pathways previously implicated in podocyte pathophysiology and is likely to sensitize these cells to other insults.

ERß regulates NSCLC phenotypes by controlling oncogenic RAS signaling.

UNLABELLED: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. In addition to the aberrant growth factor signaling, dysregulation of other pathways, such as those mediated by estrogens and their receptors, has been linked to NSCLC initiation and progression. Although the expression of wild-type estrogen receptor ß (ERß1) has been associated with prolonged disease-free survival in patients with NSCLC, the molecular mechanism that accounts for this correlation is unknown. Here, upregulation of ERß1 reduced proliferation and enhanced apoptosis in the context of mutant RAS. ERß1 was found to induce apoptosis by stimulating the intrinsic apoptotic pathway that involves BIM, a Bcl-2 proapoptotic family member that is regulated by the extracellular signal-regulated kinase (ERK). Downregulation of EGFR and inactivation of RAS and the downstream components ERK1/2 were found to be involved in the ERß1-induced apoptosis. Manipulation of EGFR and RAS expression and activity in ERß1-expressing cells revealed the central role of oncogenic RAS inhibition in the ERß1-mediated proapoptotic phenotype and EGFR regulation. These results demonstrate that ERß1 decreases the survival of NSCLC cells by regulating oncogenic RAS signaling. IMPLICATIONS: The ability of ERß1 to regulate the oncogenic functions of RAS suggests its importance in the biology of NSCLC and its clinical management. Mol Cancer Res; 12(6); 843-54. ©2014 AACR.

ERbeta1 represses basal breast cancer epithelial to mesenchymal transition by destabilizing EGFR.

INTRODUCTION: Epithelial to mesenchymal transition (EMT) is associated with the basal-like breast cancer phenotypes. 60% of basal-like cancers have been shown to express wild-type estrogen receptor beta (ERbeta1). However, it is still unclear whether the ERbeta expression is related to EMT, invasion and metastasis in breast cancer. In the present study, we examined whether ERbeta1 through regulating EMT can influence invasion and metastasis in basal-like cancers. METHODS: Basal-like breast cancer cells (MDA-MB-231 and Hs578T) in which ERbeta1 was either overexpressed or downregulated were analyzed for their ability to migrate and invade (wound-healing assay, matrigel-coated Transwell assay) as well as for the expression of EMT markers and components of the EGFR pathway (immunoblotting, RT-PCR). Coimmunoprecipitation and ubiquitylation assays were employed to examine whether ERbeta1 alters EGFR protein degradation and the interaction between EGFR and the ubiquitin ligase c-Cbl. The metastatic potential of the ERbeta1-expressing MDA-MB-231 cells was evaluated in vivo in a zebrafish xenotransplantation model and the correlation between ERbeta1 and E-cadherin expression was examined in 208 clinical breast cancer specimens by immunohistochemistry. RESULTS: Here we show that ERbeta1 inhibits EMT and invasion in basal-like breast cancer cells when they grow either in vitro or in vivo in zebrafish. The inhibition of EMT correlates with an ERbeta1-mediated upregulation of miR-200a/b/429 and the subsequent repression of ZEB1 and SIP1, which results in increased expression of E-cadherin. The positive correlation of ERbeta1 and E-cadherin expression was additionally observed in breast tumor samples. Downregulation of the basal marker EGFR through stabilization of the ubiquitin ligase c-Cbl complexes and subsequent ubiquitylation and degradation of the activated receptor is involved in the ERbeta1-mediated repression of EMT and induction of EGFR signaling abolished the ability of ERbeta1 to sustain the epithelial phenotype. CONCLUSIONS: Taken together, the results of our study strengthen the association of ERbeta1 with the regulation of EMT and propose the receptor as a potential crucial marker in predicting metastasis in breast cancer.