The scaffold protein p140Cap limits ERBB2-mediated breast cancer progression interfering with Rac GTPase-controlled circuitries

The docking protein p140Cap negatively regulates tumour cell features. Its relevance on breast cancer patient survival, as well as its ability to counteract relevant cancer signalling pathways, are not fully understood. Here we report that in patients with ERBB2-amplified breast cancer, a p140Cap-positive status associates with a significantly lower probability of developing a distant event, and a clear difference in survival. p140Cap dampens ERBB2-positive tumour cell progression, impairing tumour onset and growth in the NeuT mouse model, and counteracting epithelial mesenchymal transition, resulting in decreased metastasis formation. One major mechanism is the ability of p140Cap to interfere with ERBB2-dependent activation of Rac GTPase-controlled circuitries. Our findings point to a specific role of p140Cap in curbing the aggressiveness of ERBB2-amplified breast cancers and suggest that, due to its ability to impinge on specific molecular pathways, p140Cap may represent a predictive biomarker of response to targeted anti-ERBB2 therapies.

Survival percentage of patients with SRCIN1 gain (blue line) was compared to that of patients with SRCIN1 no gain (red line). Y-axis corresponds to the percentage of survival. p=0.012.
The patients cohort was the same showed in Figure 2A. (B) p140Cap expression in different tissues of the MMTV-p140Cap transgenic mice.
Protein extracts from distinct tissues collected from FVB WT or MMTV-p140Cap Tg female at 15 days of pregnancy, were run on 6% SDS-PAGE and stained with antibodies to p140Cap or to actin, as loading control. p140Cap transgene is expressed in mammary glands, parotid glands, lungs, and, at a minor extent, in brain. Representative images out of four different animals are shown.
(C) p140Cap expression in Tg mice does not impair mammary gland development and differentiation. Mammary glands from WT and MMTV-p140Cap transgenic mice were collected at several stages of physiologic mammary development, namely at 6 weeks (puberal period) and 12 weeks (mature virgin mammary gland) of age, 12 days of pregnancy, 3 days of lactation and 5 days of post weaning involution. Five parameters were analysed to compare WT and Tg mammary glands (see below), and reported in histograms (mean ± SEM). No differences in TEB number, ductal elongation and ductal network area during mammary gland growth and development in virgin mice, a non significant trend to lobular increase during pregnancy and lactation and a small impairment in post weaning lobular involution in Tg mice were observed. Bar: a, b, d, g, h, j 0,2 cm; c, e, i, k 300 µm; f, l 20 µm.
(D) p140Cap is expressed into the mammary epithelial compartment of the Tg mice.
Formalin-fixed paraffin-embedded fourth abdominal mammary glands of Tg mice at 3 days of lactation were analyzed by IHC with anti p140Cap antibodies, as shown in Figure 1  Note that the number of scored cases is lower than the total number of cases since: i) in some cases, individual cores detached from the slides during the manipulations; ii) clinical information was not available for all patients. In tumor tissues the IHC signals were associated with the tumor cell component and not with the adjacent or infiltrating stroma. *Only for 515 of 622 samples expression data for p140cap was available. Figure 4 3 Parameters: The fourth abdominal mammary glands were analyzed from at least five mice 4 for age group. Only whole mounts that contained the entire ductal network including the 5 primary duct and were free of mounting artifacts such as tissue folds were used for 6 subsequent image analysis. A digital photomicrograph was taken of each whole mount using 7 an Leika MZ6 stereo microscope fitted with an Nikon Coolpix color digital microscope 8 camera. Within each age group, a consistent magnification was established that allowed the 9 entire epithelial complex to be captured in a single image. For each age group, the 10 photomicrographic settings remained constant. Four different measurements were obtained 11 from each whole-mount image using Photoshop software. TEB count was performed only on 12 6 weeks of age glands.

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Ductal length (pixels) was measured by drawing and measuring a straight line caliper 14 from the most distal point of the ductal network to the nipple.

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Ductal network area (pixels) was measured from a best-fit polygon that was drawn 16 around each ductal network using the computer mouse. Each polygon was outlined without 17 indentations so as to ensure consistency across samples.

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Epithelial area (pixels), which excluded the intervening adipose tissue, was measured 19 after the epithelial structures were outlined automatically using magic wand tool.

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Epithelial density was calculated by dividing the epithelial area by ductal area.

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TEB incidence in growing glands (6 weeks of age) was determined by manually tagging 22 individual TEB with a computer-generated marker. End buds were identified as large bulbous 23 profiles located at the termini of ducts and fitted the criteria of having a cross sectional 24 diameter that was approximately twice that of the subtending duct.