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In prostate cancer, androgen receptor (AR)-targeting agents are very effective in various stages of the disease. However, therapy resistance inevitably occurs and little is known about how tumor cells adapt to bypass AR suppression. Here, we performed integrative multi-omics analyses on tissues isolated before and after 3 months of AR-targeting enzalutamide monotherapy from high-risk prostate cancer patients enrolled in a neoadjuvant clinical trial. Transcriptomic analyses demonstrated that AR inhibition drove tumors towards a neuroendocrine-like disease state. In addition, epigenomic profiling revealed massive enzalutamide-induced reprogramming of pioneer factor FOXA1 – from inactive chromatin binding sites towards active cis-regulatory elements that dictate pro-survival signals. Notably, treatment-induced FOXA1 sites were enriched for the circadian rhythm core component ARNTL. Post-treatment ARNTL levels associated with poor outcome, and ARNTL suppression decreased cell growth in vitro. Our data highlight a remarkable cistromic plasticity of FOXA1 following AR-targeted therapy, and revealed an acquired dependency on circadian regulator ARNTL, a novel candidate therapeutic target.SignificanceUnderstanding how prostate cancer cells adapt to AR-targeted interventions is critical for identifying novel drug targets to improve the clinical management of treatment-resistant disease. Our study revealed an enzalutamide-induced epigenetic plasticity towards pro-survival signaling, and uncovered circadian regulator ARNTL as an acquired vulnerability after AR inhibition, presenting a novel clinical lead for therapeutic development.

The Androgen Receptor (AR) is the key-driving transcription factor in prostate cancer, tightly controlled by epigenetic regulation. To date, most epigenetic profiling has been performed in cell lines or limited tissue samples. Here, to comprehensively study the epigenetic landscape, we perform RNA-seq with ChIP-seq for AR and histone modification marks (H3K27ac, H3K4me3, H3K27me3) in 100 primary prostate carcinomas. Integrative molecular subtyping of the five data streams revealed three major subtypes of which two were clearly TMPRSS2-ERG dictated. Importantly, we identify a third subtype with low chromatin binding and activity of AR, but with high activity of FGF and WNT signaling. While positive for neuroendocrine-hallmark genes, these tumors were copy number-neutral with low mutational burden, significantly depleted for genes characteristic of poor-outcome associated luminal B-subtype. We present a unique resource on transcriptional and epigenetic control in prostate cancer, revealing tight control of gene regulation differentially dictated by AR over three subtypes., The Androgen Receptor (AR) is the main driver of prostate cancer and functions in conjunction with chromatin modifications. Here, the authors comprehensively profile 100 primary prostate carcinomas by sequencing RNA transcripts in combination with ChIP-sequencing for AR and the active histone marks H3K27ac, H3K4me3 and repressive mark H3K27me3.

Contains fulltext : 200105.pdf (Publisher’s version ) (Open Access) Estrogen receptor-alpha (ERalpha)-positive breast cancer is often treated with antihormonal regimens. However, resistance to treatment is common, leading to metastatic disease. ERalpha activity requires the functional involvement of pioneer factors FOXA1 and GATA3, which enable ERalpha-chromatin binding and are crucial for ERalpha-driven cell proliferation. FOXA1 was found increased in metastatic breast cancers in relation to the primary tumor, but a comprehensive clinical assessment thereof, in relation to different metastatic sites and endocrine therapy usage, is currently lacking. Prior cell line-based reports, however, have revealed that FOXA1 is required for tamoxifen-resistant tumor cell proliferation. We studied expression levels of ERalpha, GATA3, and FOXA1 by immunohistochemistry in samples from both primary tumors and various metastatic sites. For all factors, expression levels varied between the metastatic sites. For pleural metastases, strong variation was found in FOXA1 and GATA3 levels. Although GATA3 levels remained unaltered between primary breast cancer and pleural metastases, FOXA1 levels were reduced exclusively in metastases of patients who received endocrine therapies in the adjuvant setting, even though ERalpha was still expressed. Importantly, decreased FOXA1 levels in pleural metastases correlated with hormone irresponsiveness in the palliative setting, while no such correlation was found for GATA3. With this, we show divergent clinical correlations of the two ERalpha pioneer factors FOXA1 and GATA3 in metastatic breast cancer, where endocrine therapy resistance was associated with decreased FOXA1 levels in pleural metastases. 01 november 2018

Background IGF-1R is overexpressed in a substantial number of breast cancer cases. When phosphorylated, IGF-1R activates the PI3K and MAPK pathways. In preclinical studies, IGF-1R overexpression has been described to be associated with tamoxifen resistance. Expression of activated IGF-1R has not been established as a marker for endocrine resistance in the clinic. We tested the value of p-IGF-1R to predict adjuvant tamoxifen benefit in IGF-1R-positive tumors from ER+ breast cancer patients and its possible association with PI3K/MAPK activation. We also carried out cell line experiments to illustrate a potential causal link between IGF-1R activation status, PI3K/MAPK pathway activation and tamoxifen resistance. Methods Primary tumor blocks from 563 ER+ (stage I-III) postmenopausal patients previously randomized between tamoxifen (1 to 3 years) vs. no adjuvant therapy (IKA trial 1982-1993; Beelen et al, Breast Cancer Res, 2014) were recollected. Immunohistochemistry scoring on tissue microarrays included PTEN, p-IGF-1R, p-AKT(Thr308), p-AKT(Ser473) and p-p70S6K(Thr389) by cytoplasmic intensity (0-3), of p-4EBP1(Ser65) and p-ERK1/2(Thr202/204) by percentage of tumor cells with positive nuclear staining and of p-S6RP(Ser235/236) by percentage of tumor cells with positive membranous staining. Informative data on p-IGF-1R staining was available for 364 IGF-1R-positive tumors. Multivariate Cox models including standard prognostic factors were used to assess hazard ratios (HR) for recurrence-free interval for tamoxifen treatment, p-IGF-1R status (negative vs. positive) and their interaction. MCF-7 and T47D cell lines were used to validate the clinical findings. IncuCyte cell proliferation experiments were performed with various IGF-1R-related growth stimulating and inhibiting conditions. Western blots were carried out on cells under various growth conditions to analyze whether activation of IGF-1R would be associated with PI3K/MAPK pathway activation. Results Patients having tumors without p-IGF-1R expression derived significant benefit from tamoxifen (HR 0.4290, 95% CI 0.2356 - 0.7813; p = 0.00566), while those having tumors with p-IGF-1R expression had no benefit (HR 0.8051, 95% CI 0.2643 – 2.453; p = 0.70). The p for interaction was not significant (p = 0.32106). Tumors positive for p-IGF-1R had more expression of (phospho)proteins downstream of the PI3K/MAPK pathways. These results were supported by Western blots from the cell lines examined under various growth conditions. In both cell lines, linsitinib (a dual IGF-1R and insulin receptor inhibitor) was able to block IGF-1R signaling, preventing activation of the PI3K and MAPK pathways and abrogating cell proliferation in the presence of tamoxifen. Conclusions Postmenopausal breast cancer patients with p-IGF-1R-positive tumors appear to derive no benefit from adjuvant tamoxifen. Tumors with p-IGF-1R expression were associated with PI3K/MAPK pathway activation. In breast cancer cell lines with activated IGF-1R signaling, addition of linsitinib to endocrine therapy can restore sensitivity. This combination might be an interesting treatment option for tamoxifen-resistant patients. Citation Format: Kruger DT, Alexi X, Opdam M, Schuurman KG, Sanders J, van der Noort V, Boven E, Zwart W, Linn SC. Phosphorylation of insulin-like growth factor-1 receptor (IGF-1R) is associated with tamoxifen resistance by activating the PI3K/MAPK pathway [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-08-06.

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., Glucocorticoids (GC) are reported to block cancer cell proliferation, but the mode of action is unclear. Here the authors show that glucocorticoid receptor activation induces cancer cell dormancy in lung cancer by regulating CDKN1C expression through a distal enhancer, and these dormant cells are addicted to IGF-1R signalling pathway.

Preclinical studies indicate that activated IGF‐1R can drive endocrine resistance in ER‐positive (ER+) breast cancer, but its clinical relevance is unknown. We studied the effect of IGF‐1R signaling on tamoxifen benefit in patients and we searched for approaches to overcome IGF‐1R‐mediated tamoxifen failure in cell lines. Primary tumor blocks from postmenopausal ER+ breast cancer patients randomized between adjuvant tamoxifen versus nil were recollected. Immunohistochemistry for IGF‐1R, p‐IGF‐1R/InsR, p‐ERα(Ser118), p‐ERα(Ser167) and PI3K/MAPK pathway proteins was performed. Multivariate Cox models were employed to assess tamoxifen efficacy. The association between p‐IGF‐1R/InsR and PI3K/MAPK pathway activation in MCF‐7 and T47D cells was analyzed with Western blots. Cell proliferation experiments were performed under various growth‐stimulating and ‐inhibiting conditions. Patients with ER+, IGF‐1R‐positive breast cancer without p‐IGF‐1R/InsR staining (n = 242) had tamoxifen benefit (HR 0.41, p = 0.0038), while the results for p‐IGF‐1R/InsR‐positive patients (n = 125) were not significant (HR 0.95, p = 0.3). High p‐ERα(Ser118) or p‐ERα(Ser167) expression was associated with less tamoxifen benefit. In MCF‐7 cells, IGF‐1R stimulation increased phosphorylation of PI3K/MAPK proteins and ERα(Ser167) regardless of IGF‐1R overexpression. This could be abrogated by the dual IGF‐1R/InsR inhibitor linsitinib, but not by the IGF‐IR‐selective antibody 1H7. In MCF‐7 and T47D cells, stimulation of the IGF‐1R/InsR pathway resulted in cell proliferation regardless of tamoxifen. Abrogation of cell growth was regained by addition of linsitinib. In conclusion, p‐IGF‐1R/InsR positivity in ER+ breast cancer is associated with reduced benefit from adjuvant tamoxifen in postmenopausal patients. In cell lines, stimulation rather than overexpression of IGF‐1R is driving tamoxifen resistance to be abrogated by linsitinib., What's new? In breast cancer, the growth factor receptor IGF‐1R is frequently overexpressed, boosting cell proliferation and transformation. Prior work has shown that IGF‐1R pathway activation promotes treatment resistance in cell lines. Here, the authors evaluated whether the pathway contributes to tamoxifen resistance in patients. In patients with ER+ breast cancer, patients whose cancers tested negative for activated IGF‐R1 benefited from adjuvant tamoxifen, while those with cancers tested positive showed little improvement. Next, they showed that treating breast cancer cells with linsitinib can thwart IGF‐1R signaling and restore tamoxifen's efficacy. Patients with activated IGF‐1R pathways may therefore do better with tamoxifen combined with linsitinib than tamoxifen alone.

Tamoxifen, a small-molecule antagonist of the transcription factor estrogen receptor alpha (ERα) used to treat breast cancer, increases risks of endometrial cancer. However, no parallels of ERα transcriptional action in breast and endometrial tumors have been found that might explain this effect. In this study, we addressed this issue with a genome-wide assessment of ERα-chromatin interactions in surgical specimens obtained from patients with tamoxifen-associated endometrial cancer. ERα was found at active enhancers in endometrial cancer cells as marked by the presence of RNA polymerase II and the histone marker H3K27Ac. These ERα binding sites were highly conserved between breast and endometrial cancer and enriched in binding motifs for the transcription factor FOXA1, which displayed substantial overlap with ERα binding sites proximal to genes involved in classical ERα target genes. Multifactorial ChIP-seq data integration from the endometrial cancer cell line Ishikawa illustrated a functional genomic network involving ERα and FOXA1 together with the enhancer-enriched transcriptional regulators p300, FOXM1, TEAD4, FNFIC, CEBP8, and TCF12. Immunohistochemical analysis of 230 primary endometrial tumor specimens showed that lack of FOXA1 and ERα expression was associated with a longer interval between breast cancer and the emergence of endometrial cancer, exclusively in tamoxifen-treated patients. Our results define conserved sites for a genomic interplay between FOXA1 and ERα in breast cancer and tamoxifen-associated endometrial cancer. In addition, FOXA1 and ERα are associated with the interval time between breast cancer and endometrial cancer only in tamoxifen-treated breast cancer patients. Cancer Res; 76(13); 3773–84. ©2016 AACR.

Male breast cancer (MBC) is rare and poorly characterized. Like the female counterpart, most MBCs are hormonally driven, but relapse after hormonal treatment is also noted. The pan-hormonal action of steroid hormonal receptors, including estrogen receptor alpha (ERα), androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) in this understudied tumor type remains wholly unexamined. This study reveals genomic cross-talk of steroid hormone receptor action and interplay in human tumors, here in the context of MBC, in relation to the female disease and patient outcome. Here we report the characterization of human breast tumors of both genders for cistromic make-up of hormonal regulation in human tumors, revealing genome-wide chromatin binding landscapes of ERα, AR, PR, GR, FOXA1, and GATA3 and enhancer-enriched histone mark H3K4me1. We integrate these data with transcriptomics to reveal gender-selective and genomic location-specific hormone receptor actions, which associate with survival in MBC patients., Male breast cancer (MBC) is rare and largely hormonally driven. Here, the authors examine the action of steroid hormone receptors in male and female breast cancers and find gender selective hormone receptor action that associates with the survival of MBC patients.

The ubiquitin proteasome system is an attractive pharmacological target for the treatment of cancer. The proteasome inhibitor bortezomib has been approved for the treatment of multiple myeloma and mantle cell lymphoma but is associated with substantial adverse effects and the occurrence of resistance, underscoring the continued need for novel proteasome inhibitors. In this study, bortezomib and the novel proteasome inhibitor delanzomib were compared for their ability to inhibit proteasome activity using both fluorogenic substrates and a recently developed fluorescent proteasome activity probe. Bortezomib and delanzomib were equipotent in inhibiting distinct subunits of the proteasome in a panel of cell lines in vitro. In a preclinical multiple myeloma model, both inhibitors inhibited the proteasome in normal tissues to a similar extent. Tumor proteasome activity was inhibited to a significantly higher extent by delanzomib (60%) compared to bortezomib (32%). In addition, delanzomib was able to overcome bortezomib resistance in vitro. The present findings demonstrate that proteasome activity probes can accurately monitor the effects of proteasome inhibitors on both normal and tumor tissues in preclinical models and can be used as a diagnostic approach to predict resistance against treatment with proteasome inhibitors. Furthermore, the data presented here provide rationale for further clinical development of delanzomib.