Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer.

BACKGROUND: Endocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER+) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility. METHODS: A panel of ER+ BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1 wt or ESR1 Y537S , modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies. RESULTS: Here, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER-mediated transactivation and ER recruitment, an effect reduced by the addition of RAD001. In-vivo analysis of an LTED model showed the triple combination of RAD001, neratinib, and fulvestrant was most effective at reducing tumour volume. Gene set enrichment analysis revealed that the addition of neratinib negated the epidermal growth factor (EGF)/EGF receptor feedback loops associated with RAD001. CONCLUSIONS: Our data support the combination of therapies targeting ERBB2/3 and mTORC1 signalling, together with fulvestrant, in patients who relapse on endocrine therapy and retain a functional ER.

Abiraterone shows alternate activity in models of endocrine resistant and sensitive disease.

BACKGROUND: Resistance to endocrine therapy remains a major clinical problem in the treatment of oestrogen-receptor positive (ER+) breast cancer. Studies show androgen-receptor (AR) remains present in 80-90% of metastatic breast cancers providing support for blockade of AR-signalling. However, clinical studies with abiraterone, which blocks cytochrome P450 17A1 (CYP17A1) showed limited benefit. METHODS: In order to address this, we assessed the impact of abiraterone on cell-viability, cell-death, ER-mediated transactivation and recruitment to target promoters. together with ligand-binding assays in a panel of ER+ breast cancer cell lines that were either oestrogen-dependent, modelling endocrine-sensitive disease, or oestrogen-independent modelling relapse on an aromatase inhibitor. The latter, harboured wild-type (wt) or naturally occurring ESR1 mutations. RESULTS: Similar to oestrogen, abiraterone showed paradoxical impact on proliferation by stimulating cell growth or death, depending on whether the cells are hormone-dependent or have undergone prolonged oestrogen-deprivation, respectively. Abiraterone increased ER-turnover, induced ER-mediated transactivation and ER-degradation via the proteasome. CONCLUSIONS: Our study confirms the oestrogenic activity of abiraterone and highlights its differential impact on cells dependent on oestrogen for their proliferation vs. those that are ligand-independent and harbour wt or mutant ESR1. These properties could impact the clinical efficacy of abiraterone in breast cancer.

Targeting Transcriptional Regulation with a CDK9 Inhibitor Suppresses Growth of Endocrine- and Palbociclib-Resistant ER+ Breast Cancers.

The combination of endocrine therapy and CDK4/6 inhibitors such as palbociclib is an effective and well-tolerated treatment for estrogen receptor-positive (ER+) breast cancer, yet many patients relapse with therapy-resistant disease. Determining the mechanisms underlying endocrine therapy resistance is limited by the lack of ability to fully recapitulate inter- and intratumor heterogeneity in vitro and of availability of tumor samples from women with disease progression or relapse. In this study, multiple cell line models of resistant disease were used for both two-dimensional (2D)- and three-dimensional (3D)-based inhibitor screening. The screens confirmed the previously reported role of pro-proliferative pathways, such as PI3K-AKT-mTOR, in endocrine therapy resistance and additionally identified the transcription-associated cyclin-dependent kinase CDK9 as a common hit in ER+ cell lines and patient-derived organoids modeling endocrine therapy-resistant disease in both the palbociclib-sensitive and palbociclib-resistant settings. The CDK9 inhibitor, AZD4573, currently in clinical trials for hematologic malignancies, acted synergistically with palbociclib in these ER+in vitro 2D and 3D models. In addition, in two independent endocrine- and palbociclib-resistance patient-derived xenografts, treatment with AZD4573 in combination with palbociclib and fulvestrant resulted in tumor regression. Tumor transcriptional profiling identified a set of transcriptional and cell-cycle regulators differentially downregulated only in combination-treated tumors. Together, these findings identify a clinically tractable combination strategy for overcoming resistance to endocrine therapy and CDK4/6 inhibitors in breast cancer and provide insight into the potential mechanism of drug efficacy in targeting treatment-resistant disease. SIGNIFICANCE: Targeting transcription-associated CDK9 synergizes with CDK4/6 inhibitor to drive tumor regression in multiple models of endocrine- and palbociclib-resistant ER+ breast cancer, which could address the challenge of overcoming resistance in patients.

Elacestrant demonstrates strong anti-estrogenic activity in PDX models of estrogen-receptor positive endocrine-resistant and fulvestrant-resistant breast cancer.

The selective oestrogen receptor (ER) degrader (SERD), fulvestrant, is limited in its use for the treatment of breast cancer (BC) by its poor oral bioavailability. Comparison of the orally bioavailable investigational SERD elacestrant, versus fulvestrant, demonstrates both drugs impact tumour growth of ER+ patient-derived xenograft models harbouring several ESR1 mutations but that elacestrant is active after acquired resistance to fulvestrant. In cell line models of endocrine sensitive and resistant breast cancer both drugs impact the ER-cistrome, ER-interactome and transcription of oestrogen-regulated genes similarly, confirming the anti-oestrogenic activity of elacestrant. The addition of elacestrant to CDK4/6 inhibitors enhances the antiproliferative effect compared to monotherapy. Furthermore, elacestrant inhibits the growth of palbociclib-resistant cells. Lastly, resistance to elacestrant involves Type-I and Type-II receptor tyrosine kinases which are amenable to therapeutic targeting. Our data support the wider clinical testing of elacestrant.

Tumour kinome re-wiring governs resistance to palbociclib in oestrogen receptor positive breast cancers, highlighting new therapeutic modalities.

Combination of CDK4/6 inhibitors and endocrine therapy improves clinical outcome in advanced oestrogen receptor (ER)-positive breast cancer, however relapse is inevitable. Here, we show in model systems that other than loss of RB1 few gene-copy number (CN) alterations are associated with irreversible-resistance to endocrine therapy and subsequent secondary resistance to palbociclib. Resistance to palbociclib occurred as a result of tumour cell re-wiring leading to increased expression of EGFR, MAPK, CDK4, CDK2, CDK7, CCNE1 and CCNE2. Resistance altered the ER genome wide-binding pattern, leading to decreased expression of 'classical' oestrogen-regulated genes and was accompanied by reduced sensitivity to fulvestrant and tamoxifen. Persistent CDK4 blockade decreased phosphorylation of tuberous sclerosis complex 2 (TSC2) enhancing EGFR signalling, leading to the re-wiring of ER. Kinome-knockdown confirmed dependency on ERBB-signalling and G2/M-checkpoint proteins such as WEE1, together with the cell cycle master regulator, CDK7. Noteworthy, sensitivity to CDK7 inhibition was associated with loss of ER and RB1 CN. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance and highlights new therapeutic networks that can be exploited upon relapse after CDK4/6 inhibition.

De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy.

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.

PLK1 inhibition exhibits strong anti-tumoral activity in CCND1-driven breast cancer metastases with acquired palbociclib resistance.

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.

Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance.

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.

Src Is a Potential Therapeutic Target in Endocrine-Resistant Breast Cancer Exhibiting Low Estrogen Receptor-Mediated Transactivation.

Despite the effectiveness of endocrine therapies in estrogen receptor positive (ER+) breast cancer, approximately 40% of patients relapse. Previously, we identified the Focal-adhesion kinase canonical pathway as a major contributor of resistance to estrogen deprivation and cellular-sarcoma kinase (c-src) as a dominant gene in this pathway. Dasatinib, a pan-src inhibitor, has recently been used in clinical trials to treat ER+ patients but has shown mixed success. In the following study, using isogenic cell line models, we provide a potential explanation for these findings and suggest a sub-group that may benefit. A panel of isogenic cell lines modelling resistance to aromatase inhibitors (LTED) and tamoxifen (TAMR) were assessed for response to dasatinib ± endocrine therapy. Dasatinib caused a dose-dependent decrease in proliferation in MCF7-TAMR cells and resensitized them to tamoxifen and fulvestrant but not in HCC1428-TAMR. In contrast, in estrogen-deprived conditions, dasatinib increased the proliferation rate of parental-MCF7 cells and had no effect on MCF7-LTED or HCC1428-LTED. Treatment with dasatinib caused a decrease in src-phosphorylation and inhibition of downstream pathways, including AKT and ERK1/2 in all cell lines tested, but only the MCF7-TAMR showed a concomitant decrease in markers of cell cycle progression. Inhibition of src also caused a significant decrease in cell migration in both MCF7-LTED and MCF7-TAMR cells. Finally, we showed that, in MCF7-TAMR cells, in contrast to tamoxifen sensitive cell lines, ER is expressed throughout the cell rather than being restricted to the nucleus and that treatment with dasatinib resulted in nuclear shuttling of ER, which was associated with an increase in ER-mediated transcription. These data suggest that src has differential effects in endocrine-resistant cell lines, particularly in tamoxifen resistant models, with low ER genomic activity, providing further evidence of the importance of patient selection for clinical trials testing dasatinib utility in ER+ breast cancer.

Partially Evoked Epithelial-Mesenchymal Transition (EMT) Is Associated with Increased TGFß Signaling within Lesional Scleroderma Skin.

The origin of myofibroblasts in fibrotic conditions remains unknown and in systemic sclerosis (SSc) it has been proposed that activation of local fibroblasts, trans-differentiation of perivascular or vascular cells, recruitment of fibrocyte progenitors, or epithelial to mesenchymal transition (EMT) could be contributing. Data from our laboratory indicate that the epidermis in scleroderma is activated with the keratinocytes exhibiting a phenotype normally associated with tissue repair, including phosphorylation profiles indicative of TGFß signaling. Since TGFß is a known inducer of EMT, we investigated if there is evidence of this process in the SSc epidermis. In order to validate antibodies and primers, EMT was modeled in HaCaT cells cultured in the presence of TGFß1. Skin sections were stained with phosho-SMAD2/3, as well as with epithelial and mesenchymal markers. Moreover, mRNA levels of transcription factors associated with EMT were studied in epidermal blister sheets. We observed critical changes in the scleroderma epidermis; showing significantly increased nuclear translocation of phosphorylated Smad2/3, consistent with active TGFß signaling in SSc keratinocytes. While profound EMT could be induced in keratinocytes in vitro with the appearance of SNAI1/2 and FSP-1, and an accompanying loss of E-cadherin, in the scleroderma skin active TGFß signaling was accompanied by only partial EMT-like changes characterised by induction of SNAI1 alone and with no loss of E-cadherin. Together, our findings support a model of altered differentiation and TGFß dependent activation of scleroderma epithelial cells leading to a partially evoked EMT like process in the fibrotic skin.

A Role of Myocardin Related Transcription Factor-A (MRTF-A) in Scleroderma Related Fibrosis.

In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix.

Abnormally differentiating keratinocytes in the epidermis of systemic sclerosis patients show enhanced secretion of CCN2 and S100A9.

Skin involvement with dermal fibrosis is a hallmark of systemic sclerosis (SSc), and keratinocytes may be critical regulators of fibroblast function through secretion of chemo-attracting agents, as well as through growth factors and cytokines influencing the phenotype and proliferation rate of fibroblasts. Epithelial-fibroblast interactions have an important role in fibrosis in general. We have characterized the SSc epidermis and asked whether SSc-injured epidermal cells release factors capable of promoting fibrosis. Our results show that the SSc epidermis is hypertrophic, and has altered expression of terminal differentiation markers involucrin, loricrin, and filaggrin. Multiplex profiling revealed that SSc epidermal explants release increased levels of CCN2 and S100A9. CCN2 induction was found to spread into the upper papillary dermis, whereas S100A9 was shown to induce fibroblast proliferation and to enhance fibroblast CCN2 expression via Toll-like receptor 4. These data suggest that the SSc epidermis provides an important source of pro-fibrotic CCN2 and proinflammatory S100A9 in SSc skin, and therefore contributes to the fibrosis and inflammation seen in the disease.

Candidatus Rickettsia hoogstraalii in Ethiopian Argas persicus ticks.

Ethiopian soft ticks Argas persicus, hard ticks including both Amblyomma variegatum and Rhipicephalus (Boophilus) spp., and fleas were collected from livestock, traditional human dwellings, and cracks and crevices of trees. They were assessed in pools for the presence of Rickettsia using PCR-based methods. The extracted tick DNA was subjected to molecular screening for Rickettsia, which revealed 50.5% of the pooled samples to be positive for Rickettsia spp. These were then subjected to multi-gene analysis using both outer surface proteins and housekeeping genes with proven discriminatory potential. Sequencing of the citrate synthase and outer membrane genes clearly led to the identification of three distinct rickettsial species, Candidatus Rickettsia hoogstraalii in Argas persicus ticks; R. africae in hard tick pools, and R. felis in fleas. Furthermore, we demonstrated the presence of the plasmid-borne small heat-shock protein gene hsp2 in DNA from A. persicus ticks suggesting that Candidatus R. hoogstraalii carried by these ticks possess a plasmid. Unlike chromosomal gene sequences, the hsp2 gene failed to cluster with Candidatus R. hoogstraalii, instead falling into an isolated separate clade, suggesting a different origin for the plasmid.