Diana Martins

Alterations in Vitamin D signalling and metabolic pathways in breast cancer progression: a study of VDR, CYP27B1 and CYP24A1 expression in benign and malignant breast lesions Vitamin D pathways unbalanced in breast lesions

BackgroundBreast cancer is a heterogeneous disease associated with different patient prognosis and responses to therapy. Vitamin D has been emerging as a potential treatment for cancer, as it has been demonstrated that it modulates proliferation, apoptosis, invasion and metastasis, among others. It acts mostly through the Vitamin D receptor (VDR) and the synthesis and degradation of this hormone are regulated by the enzymes CYP27B1 and CYP24A1, respectively. We aimed to study the expression of these three proteins by immunohistochemistry in a series of breast lesions.MethodsWe have used a cohort comprising normal breast, benign mammary lesions, carcinomas in situ and invasive carcinomas and assessed the expression of the VDR, CYP27B1 and CYP24A1 by immunohistochemistry.ResultsThe results that we have obtained show that all proteins are expressed in the various breast tissues, although at different amounts. The VDR was frequently expressed in benign lesions (93.5%) and its levels of expression were diminished in invasive tumours (56.2%). Additionally, the VDR was strongly associated with the oestrogen receptor positivity in breast carcinomas. CYP27B1 expression is slightly lower in invasive carcinomas (44.6%) than in benign lesions (55.8%). In contrast, CYP24A1 expression was augmented in carcinomas (56.0% in in situ and 53.7% in invasive carcinomas) when compared with that in benign lesions (19.0%).ConclusionsFrom this study, we conclude that there is a deregulation of the Vitamin D signalling and metabolic pathways in breast cancer, favouring tumour progression. Thus, during mammary malignant transformation, tumour cells lose their ability to synthesize the active form of Vitamin D and respond to VDR-mediated Vitamin D effects, while increasing their ability to degrade this hormone.

Synergy of combined doxycycline/TUDCA treatment in lowering Transthyretin deposition and associated biomarkers: studies in FAP mouse models.

Familial Amyloidotic Polyneuropathy (FAP) is a disorder characterized by the extracellular deposition of fibrillar Transthyretin (TTR) amyloid, with a special involvement of the peripheral nerve. We had previously shown that doxycycline administered for 3 months at 40 mg/Kg/ml in the drinking water, was capable of removing TTR amyloid deposits present in stomachs of old TTR-V30M transgenic mice; the removal was accompanied by a decrease in extracellular matrix remodeling proteins that accompany fibrillar deposition, but not of non-fibrillar TTR deposition and/or markers associated with pre-fibrillar deposits. On the other hand, Tauroursodeoxycholic acid (TUDCA), a biliary acid, administrated to the same mouse model was shown to be effective at lowering deposited non-fibrillar TTR, as well as the levels of markers associated with pre-fibrillar TTR, but only at young ages.In the present work we evaluated different doxycycline administration schemes, including different periods of treatment, different dosages and different FAP TTR V30M animal models. Evaluation included CR staining, immunohistochemistry for TTR, metalloproteinase 9 (MMP-9) and serum amyloid P component (SAP). We determined that a minimum period of 15 days of treatment with a 8 mg/Kg/day dosage resulted in fibril removal. The possibility of intermittent treatments was also assessed and a maximum period of 15 days of suspension was determined to maintain tissues amyloid-free. Combined cycled doxycycline and TUDCA administration to mice with amyloid deposition, using two different concentrations of both drugs, was more effective than either individual doxycycline or TUDCA, in significantly lowering TTR deposition and associated tissue markers. The observed synergistic effect of doxycycline/TUDCA in the range of human tolerable quantities, in the transgenic TTR mice models prompts their application in FAP, particularly in the early stages of disease.

Loss of caveolin-1 and gain of MCT4 expression in the tumor stroma : key events in the progression from an in situ to an invasive breast carcinoma

The progression from in situ to invasive breast carcinoma is still an event poorly understood. However, it has been suggested that interactions between the neoplastic cells and the tumor microenvironment may play an important role in this process. Thus, the determination of differential tumor-stromal metabolic interactions could be an important step in invasiveness. The expression of stromal Caveolin-1 (Cav-1) has already been implicated in the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Additionally, stromal Cav-1 expression has been associated with the expression of stromal monocarboxylate transporter 4 (MCT4) in invasive breast cancer. However, the role of stromal MCT4 in invasiveness has never been explored, neither the association between Cav-1 and MCT4 in the transition from breast DCIS to IDC. Therefore, our aim was to investigate in a series of breast cancer samples including matched in situ and invasive components, if there was a relationship between stromal Cav-1 and MCT4 in the progression from in situ to invasive carcinoma. We found loss of stromal Cav-1 in the progression to IDC in 75% of the cases. In contrast, MCT4 stromal expression was acquired in 87% of the IDCs. Interestingly, a concomitant loss of Cav-1 and gain of MCT4 was observed in the stroma of 75% of the cases, when matched in situ and invasive carcinomas were compared. These results suggest that alterations in Cav-1 and MCT4 may thus mark a critical point in the progression from in situ to invasive breast cancer.

Molecular phenotypes of matched in situ and invasive components of breast carcinomas

The current system of pathologic classification of human breast cancers does not take into account the biologic determinants of prognosis, nor is there a consensus regarding the progression from in situ to invasive carcinoma. The present study compared the molecular phenotypes of in situ and invasive components of breast cancer in the same sample. We built a series of 189 in situ and invasive carcinomas using tissue microarrays and classified them according to their immunoprofiles regarding estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, epidermal growth factor receptor, cytokeratin 5, P-cadherin, and the antigen Ki-67 into luminal A and B, human epidermal growth factor receptor 2 overexpressing, and basal-like carcinomas. We also correlated the subgroups of carcinomas with some of the classical prognostic factors such as histologic grade, tumor size, and lymph node metastasis, as well as with the age of the patient at diagnosis. The overall concordance on the molecular phenotypes between in situ and invasive components was 94%. For the in situ component, 63% of the cases were luminal A; 15%, luminal B; 12%, human epidermal growth factor receptor 2 overexpressing; and 7%, basal-like. Regarding the invasive component, 61% of the cases were luminal A; 16%, luminal B; 12%, human epidermal growth factor receptor 2 overexpressing; and 8%, basal-like. The present study allowed the identification of different immunoprofiles of in situ and invasive breast carcinomas using a specific panel of biomarkers and showed that in most cases, there is a concordance between in situ and invasive component profiles, supporting the theory of parallel disease in breast tumorigenesis.

Altered PPP2R2A and Cyclin D1 expression defines a subgroup of aggressive luminal-like breast cancer

BackgroundPPP2R2A deletions were recently linked to a subgroup of luminal breast carcinoma (BC) that exhibits poor survival. This subgroup also exhibited amplification of a chromosome region containing the Cyclin D1 coding gene, CCND1. Therefore, we aimed to investigate whether a combination of PPP2R2A (B55α) and Cyclin D1 expression statuses evaluated by immunohistochemistry (IHC) could define a subgroup of luminal BC that exhibits poor survival.MethodsFirst we conducted a retrospective cohort study using sequencing data from The Cancer Genome Atlas initiative to correlate PPP2R2A copy number alteration (CNA) status with its expression level and the corresponding overall survival (OS). Next, also using a retrospective cohort study design, we evaluated the PPP2R2A (B55α) expression levels by IHC in a total of 807 BC patients from two independent cohorts (discovery cohort n = 349 and validation cohort n = 458). Cyclin D1 expression was also evaluated, and the PPP2R2A (B55α)-/low/Cyclin D1high phenotype was evaluated as a predictor of disease-free survival (DFS) and OS in luminal-like BC patients.ResultsDeletions in the PPP2R2A gene strongly correlate with lower mRNA expression and poorer OS. PPP2R2A (B55α)-/low carcinomas have significantly shorter DFS and OS. Furthermore, in univariate analysis, the PPP2R2A (B55α)-/low/Cyclin D1high phenotype is significantly associated with poorer DFS and OS. In a multivariate analysis, the PPP2R2A (B55α)-/low/Cyclin D1high phenotype is significantly associated with poor DFS, thus defining a group of luminal-like BC with higher risk of relapse.ConclusionWe demonstrate that BCs harboring PPP2R2A deletions are associated with worse OS. Moreover, this is the first study to demonstrate that the combination of altered PPP2R2A (B55α) and high Cyclin D1 expression by IHC defines a subgroup of luminal-like BC patients with a high risk of relapse and death.

Metastatic breast cancer: mechanisms and opportunities for cytology.

Despite significant advances in diagnosis, surgical techniques, general patient care, and local and systemic adjuvant therapies, metastatic disease remains the most critical condition limiting the survival of patients with breast cancer. Therefore, the development of effective treatment against late‐arising metastasis has become the centre of clinical attention and is one of the current challenges in cancer research. A deeper understanding of the metastatic cascade is fundamental, and the need for repetitive tumour assessments for the evaluation of tumour evolution is a relatively new practice in routine medical care. As such, fine needle aspiration cytology (FNAC) is ideally placed to monitor biological changes in metastasis that may affect treatment and response. As FNAC is a minimally invasive method, it can be performed repeatedly with relatively little trauma, and selective ancillary tests can be applied to FNAC specimens, including for tumour whose primary nature is known. Herein, we review how the linear and parallel models explain metastatic dissemination, thus influencing therapeutic and clinical decisions, and how cytology, together with immunocytochemistry and molecular analysis, can be a tool for routine clinical practice and clinical trials aimed at metastatic disease with a special emphasis on breast cancer.

The basal epithelial marker P-cadherin associates with breast cancer cell populations harboring a glycolytic and acid-resistant phenotype

BackgroundCancer stem cells are hypoxia-resistant and present a preponderant glycolytic metabolism. These characteristics are also found in basal-like breast carcinomas (BLBC), which show increased expression of cancer stem cell markers.Recently, we demonstrated that P-cadherin, a biomarker of BLBC and a poor prognostic factor in this disease, mediates stem-like properties and resistance to radiation therapy. Thus, the aim of the present study was to evaluate if P-cadherin expression was associated to breast cancer cell populations with an adapted phenotype to hypoxia.MethodsImmunohistochemistry was performed to address the expression of P-cadherin, hypoxic, glycolytic and acid-resistance biomarkers in primary human breast carcinomas. In vitro studies were performed using basal-like breast cancer cell lines. qRT-PCR, FACS analysis, western blotting and confocal microscopy were used to assess the expression of P-cadherin after HIF-1α stabilization, achieved by CoCl2 treatment. siRNA-mediated knockdown was used to silence the expression of several targets and qRT-PCR was employed to evaluate the effects of P-cadherin on HIF-1α signaling. P-cadherin high and low breast cancer cell populations were sorted by FACS and levels of GLUT1 and CAIX were assessed by FACS and western blotting. Mammosphere forming efficiency was used to determine the stem cell activity after specific siRNA-mediated knockdown, further confirmed by western blotting.ResultsWe demonstrated that P-cadherin overexpression was significantly associated with the expression of HIF-1α, GLUT1, CAIX, MCT1 and CD147 in human breast carcinomas. In vitro, we showed that HIF-1α stabilization was accompanied by increased membrane expression of P-cadherin and that P-cadherin silencing led to a decrease of the mRNA levels of GLUT1 and CAIX. We also found that the cell fractions harboring high levels of P-cadherin were the same exhibiting more GLUT1 and CAIX expression. Finally, we showed that P-cadherin silencing significantly decreases the mammosphere forming efficiency in the same range as the silencing of HIF-1α, CAIX or GLUT1, validating that all these markers are being expressed by the same breast cancer stem cell population.ConclusionsOur results establish a link between aberrant P-cadherin expression and hypoxic, glycolytic and acid-resistant breast cancer cells, suggesting a possible role for this marker in cancer cell metabolism.

Cytological criteria to predict basal phenotype of breast carcinomas.

Breast carcinoma is a heterogeneous disease. It can be classified into phenotypes based on the expression of certain proteins, with distinct differences in prognosis. The basal phenotype is associated with worse prognosis and it still remains without specific treatment. However, there is currently no international consensus on the cytological criteria that could predict this phenotype. The purpose of the study was to evaluate the cytological criteria in fine‐needle aspiration biopsy and to identify their association with the basal phenotype of breast carcinoma. Fine‐needle aspiration biopsy specimens and tissue sections (mastectomy specimen) from 74 cases of high‐grade invasive ductal breast carcinomas were consecutively retrieved from the files of three institutions. Breast carcinomas were studied using the tissue microarray technique, being classified into phenotypes: luminal A, luminal B, HER2 overexpression, and basal. The cytological criteria for all cases were reviewed blindly by two pathologists according to five cytological criteria: cellularity, cell pattern, presence of necrosis, nucleoli, and nuclear atypia. Exact Fisher test was used to test the association between cytological criteria and the phenotypes of breast carcinoma. Necrosis was present in 64.7% of basal breast carcinomas, and 31.1% of nonbasal breast carcinomas, and that result was statistically significant, showing an odds ratio (OR) of 3.80. The basal phenotype, compared with the luminal A, showed more necrosis (OR = 6.97), present/prominent nucleoli (OR = 8.18), and cellularity more frequently (OR = 18.03). Necrosis, as well as present/prominent nucleoli and abundant cellularity are criteria more frequently associated to the basal phenotype of breast carcinoma. Diagn. Cytopathol. 2009.

p-mTOR expression is associated with better prognosis in luminal breast carcinoma

Aims Despite considerable interest in the PI3K/AKT/mTOR pathway in breast carcinomas (BC), published data reports contradictory results regarding the association of phosphorylated mammalian target of Rapamycin (p-mTOR) expression with clinico-pathological features and prognosis in BC. Here, we evaluate the main clinico-pathological associations with p-mTOR expression in BC, with focus on the different molecular subtypes. Methods In this retrospective study, 331 BC patients were included in final analysis. Outcome measures included disease-free survival (DFS) and overall survival (OS) times. Baseline data and outcome measures were compared between immunohistochemical p-mTOR expressing and non-expressing BCs. Subgroup analysis was performed to assess the effect of p-mTOR expression in the outcome for each BC molecular subtype. Results 43.8% of the tumours were positive for p-mTOR, with a significant correlation between p-mTOR expression with smaller (<2 cm) (p=0.021) and lower-grade tumours (p<0.001). Expression of p-mTOR was also associated with longer DFS (HR of 0.32, p<0.001) and OS (HR of 0.20, p<0.001). In a multivariable analysis, the HR remained significant with minimal change (HR=0.26, p=0.002 for OS; HR=0.40, p=0.002 for DFS). In subgroup analysis, luminal p-mTOR-expressing tumours demonstrated longer DFS and OS (HR 0.33, p=0.003; HR 0.20, p=0.003, respectively) independently of size, grade, lymph node status and Her-2 overexpression. Conclusions p-mTOR expression is associated with smaller, lower-grade and with luminal BC. In multivariable analysis, p-mTOR expression was associated with longer DFS and OS, independently of the size, grade and lymph node status, especially in luminal BCs.

Overexpression of Protocadherin-10 in Transthyretin-Related Familial Amyloidotic Polyneuropathy.

Overwhelming data suggest that oncogenic and neurodegenerative pathways share several altered cellular responses to insults such as oxidative stress, extracellular matrix remodeling, inflammation, or cell dyscommunication. Protocadherin-10 (Pcdh10) is an adhesion molecule found to protect against tumorigenesis and essential for axonal elongation and actin dynamics during development. Here, by using genome microarrays we identified for the first time Pcdh10 up-regulation in tissues from transgenic mouse models, cultured Schwann cells, and human samples from a familial form of peripheral neuropathy (familial amyloidotic polyneuropathy). Familial amyloidotic polyneuropathy is characterized by poor functional recovery and impaired nerve regenerative response after misfolding and deposition in the peripheral nervous system of mutant transthyretin. Not only increased transcriptional and translational Pcdh10 levels occurred in axons and Schwann cells of nerves with deposited transthyretin aggregates but the pattern also extended to associated cues of axon guidance like neuropilin-1 and F-actin. These findings suggest that Pcdh10 may influence subcellular actin cytoskeletal organization and axon-axon interactions in the course of familial amyloidotic polyneuropathy. Moreover, when preventing nonfibrillar transthyretin deposition with anakinra or transthyretin siRNA, Pcdh10 protein levels were reduced, highlighting its potential as a novel disease biomarker. Whether Pcdh10 overexpression in familial amyloidotic polyneuropathy represents a protective or deleterious response, enhancing survival or promoting cell death will need further investigation.