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

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 ESR1wt or ESR1Y537S, 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

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.Cancer Research U

Scleroderma and related disorders: 223. Long Term Outcome in a Contemporary Systemic Sclerosis Cohort

Background: We have previously compared outcome in two groups of systemic sclerosis (SSc) patients with disease onset a decade apart and we reported data on 5 year survival and cumulative incidence of organ disease in a contemporary SSc cohort. The present study examines longer term outcome in an additional cohort of SSc followed for 10 years. Methods: We have examined patients with disease onset between years 1995 and 1999 allowing for at least 10 years of follow-up in a group that has characteristics representative for the patients we see in contemporary clinical practice. Results: Of the 398 patients included in the study, 252 (63.3%) had limited cutaneous (lc) SSc and 146 (36.7%) had diffuse cutaneous (dc) SSc. The proportion of male patients was higher among the dcSSc group (17.1% v 9.9%, p = 0.037) while the mean age of onset was significantly higher among lcSSc patients (50 ± 13 v 46 ± 13 years ± SD, p = 0.003). During a 10 year follow-up from disease onset, 45% of the dcSSc and 21% of the lcSSc subjects developed clinically significant pulmonary fibrosis, p < 0.001. Among them approximately half reached the endpoint within the first 3 years (23% of dcSSc and 10% of lcSSc) and over three quarters within the first 5 years (34% and 16% respectively). There was a similar incidence of pulmonary hypertension (PH) in the two subsets with a steady rate of increase over time. At 10 years 13% of dcSSc and 15% of lcSSc subjects had developed PH (p=0.558), with the earliest cases observed within the first 2 years of disease. Comparison between subjects who developed PH in the first and second 5 years from disease onset demonstrated no difference in demographic or clinical characteristics, but 5-year survival from PH onset was better among those who developed this complication later in their disease (49% v 24%), with a strong trend towards statistical significance (p = 0.058). Incidence of SSc renal crisis (SRC) was significantly higher among the dcSSc patients (12% v 4% in lcSSc, p = 0.002). As previously observed, the rate of development of SRC was highest in the first 3 years of disease- 10% in dcSSc and 3% in lcSSc. All incidences of clinically important cardiac disease developed in the first 5 years from disease onset (7% in dcSSc v 1% in lcSSc, p < 0.001) and remained unchanged at 10 years. As expected, 10-year survival among lcSSc subjects was significantly higher (81%) compared to that of dcSSc patients (70%, p = 0.006). Interestingly, although over the first 5 years the death rate was much higher in the dcSSc cohort (16% v 6% in lcSSc), over the following years it became very similar for both subsets (14% and 13% between years 5 and 10, and 18% and 17% between years 10 and 15 for dcSSc and lcSSc respectively). Conclusions: Even though dcSSc patients have higher incidence for most organ complications compared to lcSSc subjects, the worse survival among them is mainly due to higher early mortality rate. Mortality rate after first 5 years of disease becomes comparable in the two disease subsets. Disclosure statement: The authors have declared no conflicts of interes

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

Epithelial and mesenchymal markers in SSc skin.

<p>(A) Representative images of double immunofluorescent staining of forearm skin sections performed to detect basement membrane protein collagen IV (red) and mesenchymal cell marker FSP-1 (green). The basement membrane collagen IV was not seen to be compromised in SSc, whereas some abnormal expression of FSP-1 in keratinocytes was seen in the SSc sections. (B) Additional stains for E-cadherin (green) and vimentin (red) were performed showing no loss of E-cadherin in SSc epidermal cells. Some expression of vimentin was however observed in the SSc epidermis. (C)Further analysis of FSP-1 positive cells was confirmed, since Langerhans cells within the epidermis are known to stain positive for mesenchymal markers. Immunostaining for FSP-1 (green) and Langerin (red) revealed that at least some of the FSP-1 positive cells were indeed from the Langerhans cell population (double positive cells shown with arrow). (BM = basement membrane, BV = blood vessels).</p

Canonical TGFβ signaling in SSc skin.

<p>(A) Representative images of double immunofluorescent staining performed to detect phospho-Smad2/3 (red) and K14 (green) in the epidermis of SSc patients and controls forearm skin sections (both n = 6, means of 5 high power views for each individual patient or control). DAPI (blue) was used to stain nuclei. Nuclear translocation of phospho-Smad2/3 was seen in SSc epidermal cells extending to suprabasal and granular layers. K14 expression was found to extend into suprabasal layers abnormally in SSc consistent with altered differentiation. (B) Also phospho-Smad2/3 nuclear translocation was seen in cells within the papillary dermis, increased in SSc sections (blue DAPI, red pSMAD2/3, double positive cells indicated with arrows). (C) When quantified the mean number of phospho-Smad2/3 positive cells was increased in SSc both in the epidermis (p<0.001) and in the adjacent papillary dermis (p<0.05) consistent with active TGFβ signaling. (D) qPCR of whole epidermal sheets obtained during suction blister formation revealed increased expression of SNAI1 transcription factor downstream of TGFβ. However SNAI2 was not increased. (ED = epidermis, PD = papillary dermis, RD = reticular dermis, ** = P<0.001, * = P<0.05).</p

TGFβ stimulated EMT in HaCat cells.

<p>(A) HaCat cells cultured for 72hrs with TGFβ1 4ng/ml became elongated, lost cytokeratin and induced FSP-1 expression consistent with transition to a mesenchymal phenotype. (B) Culture with TGFβ1 also led to induction of both <i>SNAI1</i> and <i>SNAI2</i> mRNA maximal with TGFβ1 2 ng/ml and consistent with fully evoked EMT.</p

Dasatinib promotes nuclear shuttling of ER in the MCF7-TAMR cells but not the 1%MCF7.

<p>1%MCF7 and MCF7-TAMR cells were treated for 24-hours with 1%FBS, dasatinib (100nM), 4-OHT (10nM), or the combination and stained with ER, pSrc^tyr416 and DAPI; bars indicate 20μm <b>(A)</b>. Schematic diagram showing cross-talk between non-genomic ER and Src. ER associates with Src at the cell membrane via a non-genomic mechanism. This leads to an increase in both ERK1/2 and AKT providing a survival advantage. The reduced genomic activity of ER in this setting enhances resistance to tamoxifen. Inhibition of Src with dasatinib causes ER to shuttle to the nucleus where it is targeted by tamoxifen, leading to a decrease in proliferation and re-sensitization to the endocrine agent <b>(B)</b>.</p