Differential transcriptional invasion signatures from patient derived organoid models define a functional prognostic tool for head and neck cancer.

Clinical outcome for patients suffering from HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. This is mostly due to highly invasive tumors that cause loco-regional relapses after initial therapeutic intervention and metastatic outgrowth. The molecular pathways governing the detrimental invasive growth modes in HNSCC remain however understudied. Here, we have established HNSCC patient derived organoid (PDO) models that recapitulate 3-dimensional invasion in vitro. Single cell mRNA sequencing was applied to study the differences between non-invasive and invasive conditions, and in a collective versus single cell invading PDO model. Differential expression analysis under invasive conditions in Collagen gels reveals an overall upregulation of a YAP-centered transcriptional program, irrespective of the invasion mode. However, we find that collectively invading HNSCC PDO cells show elevated levels of YAP transcription targets when compared to single cell invasion. Also, collectively invading cells are characterized by increased nuclear translocation of YAP within the invasive strands, which coincides with Collagen-I matrix alignment at the invasive front. Using gene set enrichment analysis, we identify immune cell-like migratory pathways in the single cell invading HNSCC PDO, while collective invasion is characterized by overt upregulation of adhesion and migratory pathways. Lastly, based on clinical head and neck cancer cohorts, we demonstrate that the identified collective invasion signature provides a candidate prognostic platform for survival in HNSCC. By uncoupling collective and single cell invasive programs, we have established invasion signatures that may guide new therapeutic options.

Proteomics on malignant pleural effusions reveals ERα loss in metastatic breast cancer associates with SGK1-NDRG1 deregulation.

Breast cancer (BCa) is a highly heterogeneous disease, with hormone receptor status being a key factor in patient prognostication and treatment decision-making. The majority of primary tumours are positive for oestrogen receptor alpha (ERα), which plays a key role in tumorigenesis and disease progression, and represents the major target for treatment of BCa. However, around one-third of patients with ERα-positive BCa relapse and progress into the metastatic stage, with 20% of metastatic cases characterised by loss of ERα expression after endocrine treatment, known as ERα-conversion. It remains unclear whether ERα-converted cancers are biologically similar to bona fide ERα-negative disease and which signalling cascades compensate for ERα loss and drive tumour progression. To better understand the biological changes that occur in metastatic BCa upon ERα loss, we performed (phospho)proteomics analysis of 47 malignant pleural effusions derived from 37 BCa patients, comparing ERα-positive, ERα-converted and ERα-negative cases. Our data revealed that the loss of ERα-dependency in this metastatic site leads to only a partial switch to an ERα-negative molecular phenotype, with preservation of a luminal-like proteomic landscape. Furthermore, we found evidence for decreased activity of several key kinases, including serum/glucocorticoid regulated kinase 1 (SGK1), in ERα-converted metastases. Loss of SGK1 substrate phosphorylation may compensate for the loss of ERα-dependency in advanced disease and exposes a potential therapeutic vulnerability that may be exploited in treating these patients.

Luminal breast cancer identity is determined by loss of glucocorticoid receptor activity.

Glucocorticoid receptor (GR) is a transcription factor that plays a crucial role in cancer biology. In this study, we utilized an in silico-designed GR activity signature to demonstrate that GR relates to the proliferative capacity of numerous primary cancer types. In breast cancer, the GR activity status determines luminal subtype identity and has implications for patient outcomes. We reveal that GR engages with estrogen receptor (ER), leading to redistribution of ER on the chromatin. Notably, GR activation leads to upregulation of the ZBTB16 gene, encoding for a transcriptional repressor, which controls growth in ER-positive breast cancer and associates with prognosis in luminal A patients. In relation to ZBTB16's repressive nature, GR activation leads to epigenetic remodeling and loss of histone acetylation at sites proximal to cancer-driving genes. Based on these findings, epigenetic inhibitors reduce viability of ER-positive breast cancer cells that display absence of GR activity. Our findings provide insights into how GR controls ER-positive breast cancer growth and may have implications for patients' prognostication and provide novel therapeutic candidates for breast cancer treatment.

Perturbations in 3D genome organization can promote acquired drug resistance.

Acquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 cells can acquire resistance to Taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here, we investigate how the ABCB1 gene is derepressed. ABCB1 activation is associated with reduced H3K9 trimethylation, increased H3K27 acetylation, and ABCB1 displacement from the nuclear lamina. While altering DNA methylation and H3K27 methylation had no major impact on ABCB1 expression, nor did it promote resistance, disrupting the nuclear lamina component Lamin B Receptor did promote the acquisition of a Taxol-resistant phenotype in a subset of cells. CRISPRa-mediated gene activation supported the notion that lamina dissociation influences ABCB1 derepression. We propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance.

Crosstalk between glucocorticoid and mineralocorticoid receptors boosts glucocorticoid-induced killing of multiple myeloma cells.

The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR action in myeloma. Here, we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that plays a role in improved myeloma cell killing. We show that the GR agonist dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells. In a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR-MR crosstalk likely arises from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR-MR heterodimerization and completely abolishes Dex-induced MR-MR homodimerization, while leaving GR-GR homodimerization intact. Unbiased transcriptomics analyses reveal that c-myc and many of its target genes are downregulated most by combined Dex-Spi treatment. Proteomics analyses further identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex-Spi downregulated genes and proteins that may predict prognosis in the CoMMpass myeloma patient cohort. Our study demonstrates that GR-MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies for glucocorticoid-based dose-reduction.

A genome-wide CRISPR screen in human prostate cancer cells reveals drivers of macrophage-mediated cell killing and positions AR as a tumor-intrinsic immunomodulator.

The crosstalk between prostate cancer (PCa) cells and the tumor microenvironment plays a pivotal role in disease progression and metastasis and could provide novel opportunities for patient treatment. Macrophages are the most abundant immune cells in the prostate tumor microenvironment (TME) and are capable of killing tumor cells. To identify genes in the tumor cells that are critical for macrophage-mediated killing, we performed a genome-wide co-culture CRISPR screen and identified AR, PRKCD, and multiple components of the NF-κB pathway as hits, whose expression in the tumor cell are essential for being targeted and killed by macrophages. These data position AR signaling as an immunomodulator, and confirmed by androgen-deprivation experiments, that rendered hormone-deprived tumor cells resistant to macrophage-mediated killing. Proteomic analyses showed a downregulation of oxidative phosphorylation in the PRKCD- and IKBKG-KO cells compared to the control, suggesting impaired mitochondrial function, which was confirmed by electron microscopy analyses. Furthermore, phosphoproteomic analyses revealed that all hits impaired ferroptosis signaling, which was validated transcriptionally using samples from a neoadjuvant clinical trial with the AR-inhibitor enzalutamide. Collectively, our data demonstrate that AR functions together with the PRKCD and the NF-κB pathway to evade macrophage-mediated killing. As hormonal intervention represents the mainstay therapy for treatment of prostate cancer patients, our findings may have direct implications and provide a plausible explanation for the clinically observed persistence of tumor cells despite androgen deprivation therapy.

PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription.

How steroid hormone receptors (SHRs) regulate transcriptional activity remains partly understood. Upon activation, SHRs bind the genome together with a co-regulator repertoire, crucial to induce gene expression. However, it remains unknown which components of the SHR-recruited co-regulator complex are essential to drive transcription following hormonal stimuli. Through a FACS-based genome-wide CRISPR screen, we functionally dissected the Glucocorticoid Receptor (GR) complex. We describe a functional cross-talk between PAXIP1 and the cohesin subunit STAG2, critical for regulation of gene expression by GR. Without altering the GR cistrome, PAXIP1 and STAG2 depletion alter the GR transcriptome, by impairing the recruitment of 3D-genome organization proteins to the GR complex. Importantly, we demonstrate that PAXIP1 is required for stability of cohesin on chromatin, its localization to GR-occupied sites, and maintenance of enhancer-promoter interactions. In lung cancer, where GR acts as tumor suppressor, PAXIP1/STAG2 loss enhances GR-mediated tumor suppressor activity by modifying local chromatin interactions. All together, we introduce PAXIP1 and STAG2 as novel co-regulators of GR, required to maintain 3D-genome architecture and drive the GR transcriptional programme following hormonal stimuli.

Enhancer profiling identifies epigenetic markers of endocrine resistance and reveals therapeutic options for metastatic castration-resistant prostate cancer patients.

Androgen Receptor (AR) signaling inhibitors, including enzalutamide, are treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC), but resistance inevitably develops. Using metastatic samples from a prospective phase II clinical trial, we epigenetically profiled enhancer/promoter activities with H3K27ac chromatin immunoprecipitation followed by sequencing, before and after AR-targeted therapy. We identified a distinct subset of H3K27ac-differentially marked regions that associated with treatment responsiveness. These data were successfully validated in mCRPC patient-derived xenograft models (PDX). In silico analyses revealed HDAC3 as a critical factor that can drive resistance to hormonal interventions, which we validated in vitro . Using cell lines and mCRPC PDX tumors in vitro , we identified drug-drug synergy between enzalutamide and the pan-HDAC inhibitor vorinostat, providing therapeutic proof-of-concept. These findings demonstrate rationale for new therapeutic strategies using a combination of AR and HDAC inhibitors to improve patient outcome in advanced stages of mCRPC.

The Tumor Coagulome as a Transcriptional Target and a Potential Effector of Glucocorticoids in Human Cancers.

BACKGROUND: The coagulome, defined as the repertoire of genes that locally regulate coagulation and fibrinolysis, is a key determinant of vascular thromboembolic complications of cancer. In addition to vascular complications, the coagulome may also regulate the tumor microenvironment (TME). Glucocorticoids are key hormones that mediate cellular responses to various stresses and exert anti-inflammatory effects. We addressed the effects of glucocorticoids on the coagulome of human tumors by investigating interactions with Oral Squamous Cell Carcinoma, Lung Adenocarcinoma, and Pancreatic Adenocarcinoma tumor types. METHODS: We analyzed the regulation of three essential coagulome components, i.e., the tissue factor (TF), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1) in cancer cell lines exposed to specific agonists of the glucocorticoid receptor (GR) (dexamethasone and hydrocortisone). We used QPCR, immunoblots, small-interfering RNA, Chromatin immunoprecipitation sequencing (ChIPseq) and genomic data from whole tumor and single-cell analyses. RESULTS: Glucocorticoids modulate the coagulome of cancer cells through a combination of indirect and direct transcriptional effects. Dexamethasone directly increased PAI-1 expression in a GR-dependent manner. We confirmed the relevance of these findings in human tumors, where high GR activity/high SERPINE1 expression corresponded to a TME enriched in active fibroblasts and with a high TGF-ß response. CONCLUSION: The transcriptional regulation of the coagulome by glucocorticoids that we report may have vascular consequences and account for some of the effects of glucocorticoids on the TME.

Mammalian life depends on two distinct pathways of DNA damage tolerance.

DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.

Ribociclib Induces Broad Chemotherapy Resistance and EGFR Dependency in ESR1 Wildtype and Mutant Breast Cancer.

While endocrine therapy is highly effective for the treatment of oestrogen receptor-α (ERα)-positive breast cancer, a significant number of patients will eventually experience disease progression and develop treatment-resistant, metastatic cancer. The majority of resistant tumours remain dependent on ERα-action, with activating ESR1 gene mutations occurring in 15-40% of advanced cancers. Therefore, there is an urgent need to discover novel effective therapies that can eradicate cancer cells with aberrant ERα and to understand the cellular response underlying their action. Here, we evaluate the response of MCF7-derived, CRISPR-Cas9-generated cell lines expressing mutant ERα (Y537S) to a large number of drugs. We report sensitivity to numerous clinically approved inhibitors, including CDK4/6 inhibitor ribociclib, which is a standard-of-care therapy in the treatment of metastatic ERα-positive breast cancer and currently under evaluation in the neoadjuvant setting. Ribociclib treatment induces senescence in both wildtype and mutant ERα breast cancer models and leads to a broad-range drug tolerance. Strikingly, viability of cells undergoing ribociclib-induced cellular senescence is maintained via engagement of EGFR signalling, which may be therapeutically exploited in both wildtype and mutant ERα-positive breast cancer. Our study highlights a wide-spread reduction in sensitivity to anti-cancer drugs accompanied with an acquired vulnerability to EGFR inhibitors following CDK4/6 inhibitor treatment.

Glucocorticoid receptor triggers a reversible drug-tolerant dormancy state with acquired therapeutic vulnerabilities in lung cancer.

The glucocorticoid receptor (GR) regulates gene expression, governing aspects of homeostasis, but is also involved in cancer. Pharmacological GR activation is frequently used to alleviate therapy-related side-effects. While prior studies have shown GR activation might also have anti-proliferative action on tumours, the underpinnings of glucocorticoid action and its direct effectors in non-lymphoid solid cancers remain elusive. Here, we study the mechanisms of glucocorticoid response, focusing on lung cancer. We show that GR activation induces reversible cancer cell dormancy characterised by anticancer drug tolerance, and activation of growth factor survival signalling accompanied by vulnerability to inhibitors. GR-induced dormancy is dependent on a single GR-target gene, CDKN1C, regulated through chromatin looping of a GR-occupied upstream distal enhancer in a SWI/SNF-dependent fashion. These insights illustrate the importance of GR signalling in non-lymphoid solid cancer biology, particularly in lung cancer, and warrant caution for use of glucocorticoids in treatment of anticancer therapy related side-effects.

Duality of glucocorticoid action in cancer: tumor-suppressor or oncogene?

Glucocorticoid receptor (GR) is a key homeostatic regulator involved in governing immune response, neuro-integration, metabolism and lung function. In conjunction with its pivotal role in human biology, GR action is critically linked to the pathology of various disease types, including cancer. While pharmacological activation of GR has been used for the treatment of various liquid cancers, its role in solid cancers is less clearly defined and seems to be cancer-type dependent. This review focuses on the molecular aspects of GR biology, spanning the structural and functional basis of response to glucocorticoids, as well as how this transcription factor operates in cancer, including the implications in disease development, progression and drug resistance.

Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites.

The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chromatin and sequence suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the result of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

Estrogen Receptor on the move: Cistromic plasticity and its implications in breast cancer.

Estrogen Receptor (ERα) is a hormone-driven transcription factor, critically involved in driving tumor cell proliferation in the vast majority of breast cancers (BCas). ERα binds the genome at cis-regulatory elements, dictating the expression of a large spectrum of responsive genes in 3D genomic space. While initial reports described a rather static ERα chromatin binding repertoire, we now know that ERα DNA interactions are highly versatile, altered in breast tumor development and progression, and deviate between tumors from patients with differential outcome. Multiple cellular signaling cascades are known to impinge on ERα genomic function, changing its cistrome to retarget the receptor to other regions of the genome and reprogram its impact on breast cell biology. This review describes the current state-of-the-art on which factors manipulate the ERα cistrome and how this alters the response to both endogenous and exogenous hormonal stimuli, ultimately impacting BCa cell progression and response to commonly used therapeutic interventions. Novel insights in ERα cistrome dynamics may pave the way for better patient diagnostics and the development of novel therapeutic interventions, ultimately improving cancer care and patient outcome.

Endonuclease FEN1 Coregulates ERα Activity and Provides a Novel Drug Interface in Tamoxifen-Resistant Breast Cancer.

Estrogen receptor α (ERα) is a key transcriptional regulator in the majority of breast cancers. ERα-positive patients are frequently treated with tamoxifen, but resistance is common. In this study, we refined a previously identified 111-gene outcome prediction-classifier, revealing FEN1 as the strongest determining factor in ERα-positive patient prognostication. FEN1 levels were predictive of outcome in tamoxifen-treated patients, and FEN1 played a causal role in ERα-driven cell growth. FEN1 impacted the transcriptional activity of ERα by facilitating coactivator recruitment to the ERα transcriptional complex. FEN1 blockade induced proteasome-mediated degradation of activated ERα, resulting in loss of ERα-driven gene expression and eradicated tumor cell proliferation. Finally, a high-throughput 465,195 compound screen identified a novel FEN1 inhibitor, which effectively blocked ERα function and inhibited proliferation of tamoxifen-resistant cell lines as well as ex vivo-cultured ERα-positive breast tumors. Collectively, these results provide therapeutic proof of principle for FEN1 blockade in tamoxifen-resistant breast cancer. SIGNIFICANCE: These findings show that pharmacologic inhibition of FEN1, which is predictive of outcome in tamoxifen-treated patients, effectively blocks ERα function and inhibits proliferation of tamoxifen-resistant tumor cells.

TLE3 loss confers AR inhibitor resistance by facilitating GR-mediated human prostate cancer cell growth.

Androgen receptor (AR) inhibitors represent the mainstay of prostate cancer treatment. In a genome-wide CRISPR-Cas9 screen using LNCaP prostate cancer cells, loss of co-repressor TLE3 conferred resistance to AR antagonists apalutamide and enzalutamide. Genes differentially expressed upon TLE3 loss share AR as the top transcriptional regulator, and TLE3 loss rescued the expression of a subset of androgen-responsive genes upon enzalutamide treatment. GR expression was strongly upregulated upon AR inhibition in a TLE3-negative background. This was consistent with binding of TLE3 and AR at the GR locus. Furthermore, GR binding was observed proximal to TLE3/AR-shared genes. GR inhibition resensitized TLE3KO cells to enzalutamide. Analyses of patient samples revealed an association between TLE3 and GR levels that reflected our findings in LNCaP cells, of which the clinical relevance is yet to be determined. Together, our findings reveal a mechanistic link between TLE3 and GR-mediated resistance to AR inhibitors in human prostate cancer.

Drivers of AR indifferent anti-androgen resistance in prostate cancer cells.

Inhibition of the androgen receptor (AR) by second-generation anti-androgens is a standard treatment for metastatic castration resistant prostate cancer (mCRPC), but it inevitably leads to the development of resistance. Since the introduction of highly efficient AR signalling inhibitors, approximately 20% of mCRPC patients develop disease with AR independent resistance mechanisms. In this study, we generated two anti-androgen and castration resistant prostate cancer cell models that do not rely on AR activity for growth despite robust AR expression (AR indifferent). They are thus resistant against all modern AR signalling inhibitors. Both cell lines display cross-resistance against the chemotherapeutic drug docetaxel due to MCL1 upregulation but remain sensitive to the PARP inhibitor olaparib and the pan-BCL inhibitor obatoclax. RNA-seq analysis of the anti-androgen resistant cell lines identified hyper-activation of the E2F cell-cycle master regulator as driver of AR indifferent growth, which was caused by deregulation of cyclin D/E, E2F1, RB1, and increased Myc activity. Importantly, mCRPC tissue samples with low AR activity displayed the same alterations and increased E2F activity. In conclusion, we describe two cellular models that faithfully mimic the acquisition of a treatment induced AR independent phenotype that is cross-resistant against chemotherapy and driven by E2F hyper-activation.