Several data support a central role for angiogenesis in breast cancer growth and metastasis. Observational studies have demonstrated that microvascular density (MVD) is a prognostic factor in invasive breast cancer, whereas others reached the opposite conclusion. Vascular endothelial growth factor is the most important angiogenic factor with proven significance in breast cancer, as it has been assessed in both experimental and clinical studies. Triple-negative breast cancer (TNBC) is a type of breast cancer which lacks estrogen, progesterone, and HER-2/neu receptors. MVD in both basal-like and TNBC is significantly higher than in non-basal-like and non-TNBC. In breast cancer and other malignancies, the development of agents that inhibit tumor angiogenesis has been an active area of investigation. In TNBC, clinical trials combining targeted agents and chemotherapy have failed to show substantial survival improvement. There is evidence that patients with TNBC may have a greater probability of obtaining some kind of clinical efficacy benefit from bevacizumab-based therapy.

The antineoplastic effect of paclitaxel is mainly related to its ability to bind the β subunit of tubulin, thus preventing tubulin chain depolarization and inducing apoptosis. The relevance of the Class I β‐tubulin characteristics have also been confirmed in the clinical setting where mutations of paclitaxel‐binding site of β‐tubulin Class I have been related to paclitaxel resistance in non small cell lung and ovarian cancers. In the present study, we verified the hypothesis of a relationship between molecular alterations of β‐tubulin Class I and paclitaxel sensitivity in a panel of breast cell lines with different drug IC₅₀. The Class I β‐tubulin gene cDNA has been sequenced detecting heterozygous missense mutations (exon 1 and 4) only in MCF‐7 and SK‐BR‐3 lines. Furthermore, the expression (at both mRNA and protein level) of the different isotypes have been analyzed demonstrating an association between low cell sensitivity to paclitaxel and Class III β‐tubulin expression increasing. Antisense oligonucleotide (ODN) experiments confirmed that the inhibition of Class III β‐tubulin could at least partially increase paclitaxel‐chemosensitivity. The hypothesis of a relationship between β‐tubulin tumor expression and paclitaxel clinical response has been finally verified in a series of 92 advanced breast cancer patients treated with a first line paclitaxel‐based chemotherapy. Thirty‐five percent (95% CI: 45–31) of patients with high Class III β‐tubulin expression showed a disease progression vs. only 7% of patients with low expression (35% vs. 7%, p < 0.002). Our study suggests that Class III β‐tubulin tumor expression could be considered a predictive biomarker of paclitaxel‐clinical resistance for breast cancer patients.

Background: Several studies looked for tumor biomarkers predictive for paclitaxel sensitivity but till now no reliable biomarkers are available. The aim of this study was to verify the potential predictive value of β-tubulin III and IV, vascular endothelial growth factor-receptor (VEGFR-1), HER2/neu and microvessel density (mVD), in a group of 70 patients with advanced breast cancer (ABC) treated with paclitaxel-based chemotherapy. Patients and methods: Immunohistochemical analysis (ICA) of HER2, VEGFR-1, mVd, β-tubulin III and β-tubulin IV expression were performed in a series of 72 advanced breast cancer. Furthermore apoptotic fraction with TUNEL analysis was evaluated. Results: β–tubulin III ICA expression was predictive of progression after chemotherapy. In fact only 2% of the patients with low β-tubulin III expression progressed after paclitaxel chemotherapy vs 38% of those with high β-tubulin III tumor expression (P=0.000; by χ²). This evidence was confirmed by a logistic regression analysis (OR 28.789; 95% CI 3.212–258,072; P=0.004). There was not a significant association between other biomarkers' characteristics and clinical response to chemotherapy. A Cox multivariate analysis, with overall survival as a dependent variable, showed that only HER2 expression was independently associated (OR 2.39; 95% CI 1.09–5.23; P=0.03) with overall survival. Conclusions: We suggest that class III β-tubulin immunohistochemical expression analysis could be a potentially relevant tumor biomarker for paclitaxel resistance in advanced breast cancer patients.

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na⁺/H⁺exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1α expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na⁺/H⁺exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.

Tryptase(+) mast cells (MCs), abundant in the invasive front of tumours, contribute to tissue remodelling. Indeed, protease‐activated receptor‐2 (PAR‐2) activation byMC‐tryptase is considered an oncogenic event in colorectal cancer (CRC). Recently, we have suggestedNHERF1 as a potential new marker inCRC. In this study, we aimed to determine the distribution of tryptase(+)MCs andPAR‐2 and to examine the relationship betweenPAR‐2 andNHERF1, investigating their reputed usefulness as tumour markers. We studied a cohort of 115CRCspecimens including primary cancer (C) and adjacent normal mucosa (NM) by immunohistochemical double staining, analyzing the protein expression ofMC‐tryptase,PAR‐2 and cytoplasmicNHERF1.MCdensity was higher inNMthan in C. Tumours with highTNMstage and poor grade showed the highestMCdensity. A higherPAR‐2 immunoreactivity characterized tumours most infiltrated byMCs compared with samples with lowMCdensity. Furthermore,PAR‐2 overexpression was associated with advancedTNMstage, poor grade and lymphovascular invasion (LVI). A positive correlation existed between tryptase(+)MCdensity andPAR‐2 expression. CytoplasmicNHERF1 was higher in C than inNMand overexpressing tumours resulted associated with nodal and distant metastases, poor grade andLVI.PAR‐2 correlated with cytoplasmicNHERF1 and thePAR‐2(+)/cytoplasmicNHERF1(+) expression immunophenotype identified tumours associated with unfavourable prognosis and aggressive clinical parameters. Our data indicate that the high density of tryptase(+)MCs at invasive margins of tumours was associated with advanced stages ofCRCand was strongly correlated withPAR‐2 expression.