Tumour-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression.

Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.

Statin-induced anti-proliferative effects via cyclin D1 and p27 in a window-of-opportunity breast cancer trial.

PURPOSE: Cholesterol lowering statins have been demonstrated to exert anti-tumoral effects on breast cancer by decreasing proliferation as measured by Ki67. The biological mechanisms behind the anti-proliferative effects remain elusive. The aim of this study was to investigate potential statin-induced effects on the central cell cycle regulators cyclin D1 and p27. EXPERIMENTAL DESIGN: This phase II window-of-opportunity trial (Trial registration: ClinicalTrials.gov NCT00816244 , NIH) included 50 patients with primary invasive breast cancer. High-dose atorvastatin (80 mg/day) was prescribed to patients for two weeks prior to surgery. Paired paraffin embedded pre- and post-statin treatment tumor samples were analyzed using immunohistochemistry for the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and the cell cycle regulators cyclin D1 and p27. Corresponding frozen tumor sample pairs were analyzed for expression of the genes coding for cyclin D1 and p27, CCND1 and CDKN1B, respectively. RESULTS: Forty-two patients completed all study parts, and immunohistochemical evaluation of ER and PR was achievable in 30 tumor pairs, HER2 in 29 tumor pairs, cyclin D1 in 30 tumor pairs and p27 in 33 tumor pairs. The expression of ER, PR and HER2 did not change significantly following atorvastatin treatment. Cyclin D1 expression in terms of nuclear intensity was significantly decreased (P = 0.008) after statin treatment in paired tumor samples. The protein expression of the tumor suppressor p27, evaluated either as the fraction of stained tumor cells or as cytoplasmic intensity, increased significantly (P = 0.03 and P = 0.02, respectively). At the transcriptional level, no significant differences in mRNA expression were detected for cyclin D1 (CCND1) and p27 (CDKN1B). However, CCND1 expression was lower in tumors responding to atorvastatin treatment with a decrease in proliferation although not significantly (P = 0.08). CONCLUSIONS: We have previously reported statin-induced anti-proliferative effects in breast cancer. This study suggests that cell cycle regulatory effects may contribute to these anti-proliferative effects via cyclin D1 and p27.

Targeting HMG-CoA reductase with statins in a window-of-opportunity breast cancer trial.

Lipophilic statins purportedly exert anti-tumoral effects on breast cancer by decreasing proliferation and increasing apoptosis. HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway, is the target of statins. However, data on statin-induced effects on HMGCR activity in cancer are limited. Thus, this pre-operative study investigated statin-induced effects on tumor proliferation and HMGCR expression while analyzing HMGCR as a predictive marker for statin response in breast cancer treatment. The study was designed as a window-of-opportunity trial and included 50 patients with primary invasive breast cancer. High-dose atorvastatin (i.e., 80 mg/day) was prescribed to patients for 2 weeks before surgery. Pre- and post-statin paired tumor samples were analyzed for Ki67 and HMGCR immunohistochemical expression. Changes in the Ki67 expression and HMGCR activity following statin treatment were the primary and secondary endpoints, respectively. Up-regulation of HMGCR following atorvastatin treatment was observed in 68 % of the paired samples with evaluable HMGCR expression (P = 0.0005). The average relative decrease in Ki67 expression following atorvastatin treatment was 7.6 % (P = 0.39) in all paired samples, whereas the corresponding decrease in Ki67 expression in tumors expressing HMGCR in the pre-treatment sample was 24 % (P = 0.02). Furthermore, post-treatment Ki67 expression was inversely correlated to post-treatment HMGCR expression (rs = -0.42; P = 0.03). Findings from this study suggest that HMGCR is targeted by statins in breast cancer cells in vivo, and that statins may have an anti-proliferative effect in HMGCR-positive tumors. Future studies are needed to evaluate HMGCR as a predictive marker for the selection of breast cancer patients who may benefit from statin treatment.

Estrogen receptor-positive breast cancer and adverse outcome in BRCA2 mutation carriers and young non-carrier patients.

Estrogen receptor-positive (ER+) breast cancer generally confers a more favorable prognosis than ER-negative cancer, however, a different picture is emerging for BRCA2 mutation carriers and young patients. We used nationwide data from population-based registries to study prognostic effects in those two groups. Of all 2817 eligible women diagnosed with breast cancer in Iceland during 1980-2004, 85% had been tested for the Icelandic 999del5 BRCA2 (c.771_775delTCAAA) founder pathogenic variant. We compared breast cancer-specific survival, effects of ER status, other clinical parameters, and treatment, between three mutually exclusive groups: BRCA2-carriers, non-carriers diagnosed 40 years or younger, and older non-carriers. Prevalence of the BRCA2 mutation among tested patients <=40 years of age was 21.0%, but it was 5.4% among women diagnosed >40 years of age. For ER+ cancer, breast cancer-specific 15-year survival was 49.7%, 55.2%, and 74.7%, among BRCA2-carriers, young and older non-carriers, respectively, whereas for ER-negative cancer, survival was similar (64.0-69.3%) for all three groups. Neither BRCA2 carriers nor young non-carriers did tumor grade 3 predict worse survival than did tumor grade 1. The adverse outcome for the young cases cannot be explained by BRCA2 mutations, as carriers were excluded from the group. Those two clinically important patient groups need special attention with respect to treatment choices, in particular, if diagnosed with ER+ tumors. It is thus advisable to have knowledge of BRCA2 status when treatment decisions are made. Finally, it is important to understand the biological basis for the specific nature of ER+ tumors in young women and BRCA2 carriers.

Incidence of and risk factors for medication-related osteonecrosis of the jaw in women with breast cancer with bone metastasis: a population-based study.

OBJECTIVE: The aim of this study was to prospectively determine the incidence of medication-related osteonecrosis of the jaw (MRONJ) and define risk factors in patients with metastatic breast cancer treated with zoledronic acid and/or denosumab. STUDY DESIGN: In a prospective cohort study performed in Region Skåne, Sweden, from January 1, 2012, until December 31, 2015, all patients with breast cancer who had radiographic evidence of bone metastases and were treated with zoledronic acid or denosumab were included and followed up until May 31, 2018. RESULTS: Of the 242 patients, MRONJ developed in 16 (6.6%) during the 77 months of study. The incidence of MRONJ in patients treated with zoledronic acid was 4.1%, and in patients treated with denosumab, it was 13.6%. The risk of MRONJ was higher in patients on denosumab than in those treated with zoledronic acid (P = .011). Corticosteroid use was associated with a decreased risk of MRONJ (P = .008), and diabetes was associated with an increased risk of MRONJ (P = .02). CONCLUSIONS: The incidence of MRONJ is 13.6% (>3 times higher) in denosumab-treated patients with breast cancer compared with that in patients treated with zoledronic acid (4.1%). Corticosteroid use decreased the risk of MRONJ.

Statin use, HMGCR expression, and breast cancer survival - The Malmö Diet and Cancer Study.

Statins, commonly used to treat hypercholesterolemia, have also been proposed as anti-cancer agents. The identification of a predictive marker is essential. The 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR), which is inhibited by statins, might serve as such a marker. Thorough antibody validation was performed for four different HMGCR antibodies. Tumor expression of HMGCR (#AMAb90619, CL0260, Atlas Antibodies, Stockholm, Sweden) was evaluated in the Malmö Diet and Cancer Study breast cancer cohort. Statin use and cause of death data were retrieved from the Swedish Prescribed Drug Register and Swedish Death Registry, respectively. Breast cancer-specific mortality (BCM) according to statin use and HMGCR expression were analyzed using Cox regression models. Three-hundred-twelve of 910 breast cancer patients were prescribed statins; 74 patients before and 238 after their breast cancer diagnosis. HMGCR expression was assessable for 656 patients; 119 showed negative, 354 weak, and 184 moderate/strong expressions. HMGCR moderate/strong expression was associated with prognostically adverse tumor characteristics as higher histological grade, high Ki67, and ER negativity. HMGCR expression was not associated with BCM. Neither was statin use associated with BCM in our study. Among breast cancer patients on statins, no or weak HMGCR expression predicted favorable clinical outcome. These suggested associations need further testing in larger cohorts.

Global Transcriptional Changes Following Statin Treatment in Breast Cancer.

BACKGROUND: Statins purportedly exert antitumoral effects, but the underlying mechanisms are currently not fully elucidated. The aim of this study was to explore potential statin-induced effects on global gene expression profiles in primary breast cancer. EXPERIMENTAL DESIGN: This window-of-opportunity phase II trial enrolled 50 newly diagnosed breast cancer patients prescribed atorvastatin (80 mg/day) for 2 weeks presurgically. Pre- and posttreatment tumor samples were analyzed using Significance Analysis of Microarrays (SAM) to identify differentially expressed genes. Similarly, SAM and gene ontology analyses were applied to gene expression data derived from atorvastatin-treated breast cancer cell lines (MCF7, BT474, SKBR3, and MDAMB231) comparing treated and untreated cells. The Systematic Motif Analysis Retrieval Tool (SMART) was used to identify enriched transcription factor-binding sites. Literature Vector Analysis (LitVAn) identified gene module functionality, and pathway analysis was performed using GeneGo Pathways Software (MetaCore; https://portal.genego.com/). RESULTS: Comparative analysis of gene expression profiles in paired clinical samples revealed 407 significantly differentially expressed genes (FDR = 0); 32 upregulated and 375 downregulated genes. Restricted filtration (fold change ≥1.49) resulted in 21 upregulated and 46 downregulated genes. Significantly upregulated genes included DUSP1, RHOB1, GADD45B, and RGS1. Pooled results from gene ontology, LitVAn and SMART analyses identified statin-induced effects on the apoptotic and MAPK pathways among others. Comparative analyses of gene expression profiles in breast cancer cell lines showed significant upregulation of the mevalonate and proapoptotic pathways following atorvastatin treatment. CONCLUSIONS: We report potential statin-induced changes in global tumor gene expression profiles, indicating MAPK pathway inhibition and proapoptotic events.