Stathmin expression associates with vascular and immune responses in aggressive breast cancer subgroups

Studies indicate that stathmin expression associates with PI3K activation in breast cancer, suggesting stathmin as a marker for targetable patient subgroups. Here we assessed stathmin in relation to tumour proliferation, vascular and immune responses, BRCA1 germline status, basal-like differentiation, clinico-pathologic features, and survival. Immunohistochemical staining was performed on breast cancers from two series (cohort 1, n = 187; cohort 2, n = 198), and mass spectrometry data from 24 cases and 12 breast cancer cell lines was examined for proteomic profiles. Open databases were also explored (TCGA, METABRIC, Oslo2 Landscape cohort, Cancer Cell Line Encyclopedia). High stathmin expression associated with tumour proliferation, p53 status, basal-like differentiation, BRCA1 genotype, and high-grade histology. These patterns were confirmed using mRNA data. Stathmin mRNA further associated with tumour angiogenesis, immune responses and reduced survival. By logistic regression, stathmin protein independently predicted a BRCA1 genotype (OR 10.0, p = 0.015) among ER negative tumours. Cell line analysis (Connectivity Map) implied PI3K inhibition in tumours with high stathmin. Altogether, our findings indicate that stathmin might be involved in the regulation of tumour angiogenesis and immune responses in breast cancer, in addition to tumour proliferation. Cell data point to potential effects of PI3K inhibition in tumours with high stathmin expression.

detail elsewhere 17,18 . After enzymatic digestion of proteins, the resulting peptides were desalted and cleaned using Oasis HLB Elution plates (Waters, Milford, MA, USA).
The retrieved peptides were separated by high-pressure liquid chromatography (HPLC) during a biphasic acetonitrile (ACN) gradient and analysed by mass spectrometry: Cell culture lysate on an LTQ-Orbitrap Velos Pro (Thermo Scientific, Bremen, Germany) with a 90 min HPLC gradient; microdissected tumour epithelium from patient tissue on a Q-Exactive HF (Thermo Fisher Scientific, Waltham, MA, USA) with a 180 min gradient.

LC-MS/MS settings used for analysis of microdissected breast cancer tumour cells.
The microdissected samples were analysed in its entirety on a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) connected to a Dionex Ultimate NCR-3500RS LC system. Samples were dissolved in 2% ACN/0.1% formic acid (FA) and trapped on the pre-column (Dionex, Acclaim PepMap 100, 2 cm x 75 µm i.d, 3 µm C18 beads) in loading buffer (0.1% trifluoroacetic acid) at a flowrate of 5 µl/min for 5 minutes, before separation by reverse phase chromatography (PepMap RSLC, 25cm x 75 µm i.d. EASY-spray column, packed with 2µm C18 beads) at a flow of 200 nL/min. Solvent A and B were 0.1% FA (vol/vol) in water and 100% ACN, respectively. The gradient composition was 5% B from 0-5 minutes, which increased linearly to 8 % from 5-5.5 minutes, to 24 % from 5.5-115 minutes, to 35 % B from 115-140 minutes and to 90 % B from 140-155 min. Washing and conditioning of the column were performed from 155-170 minutes with 90 % B, and reduced to 5% B from 170-180 minutes. The MS instrument was equipped with an EASY-spray ion source (Thermo Fisher Scientific, Waltham, MA, USA) and was operated in data-dependentacquisition mode. Instrument control was performed using Q-Exactive HF Tune 2.4 and Xcalibur 3.0. MS spectra were acquired in the scan range 375 -1500 m/z with resolution R = 120,000 at m/z 200, with an automatic gain control (AGC) target of 3e6 and a maximum injection time (IT) of 100ms. The 12 most intense eluting peptides above intensity threshold 5E4, with charge states 2 or larger, were sequentially isolated to a target AGC value of 1e5, with resolution R = 30,000, an IT of 110 ms and a normalized collision energy of 28 %. The isolation window was set to 1.6 m/z with an isolation offset of 0.3 and a dynamic exclusion of 25 seconds. Lock-mass internal calibration was used.

Gene expression data analyses
mRNA signatures. A gene expression (mRNA) signature was previously associated with increased microvessel proliferation (proliferative microvessel density; pMVD) in endometrial carcinoma 19 . This signature was associated with aggressive tumour features and reduced survival. Twenty-six of the 32 genes in the vascular proliferation score mapped to the TCGA and METABRIC breast cancer data sets. The resulting 26gene vascular proliferation scores were generated by subtracting the sum of genes down-regulated in VP-high cases from the sum of genes up-regulated in the same group, as previously described 19 . Also, scores of nestin 11

Supplementary Figure S1. Stathmin mRNA expression correlates with stemness.
Correlation between stathmin mRNA and a luminal progenitor signature score (a), a mature luminal signature score (b) and a nestin signature score (c) in the TCGA cohort.
Genes differentially expressed between stathmin -high and -low cases (sorted alphabetically).