Leticia Campo

Expression of the Hypoxia-Inducible and Tumor-Associated Carbonic Anhydrases in Ductal Carcinoma in Situ of the Breast

Carbonic anhydrases (CA) influence intra- and extracellular pH and ion transport in varied biological processes. We recently identified CA9 and CA12 as hypoxia-inducible genes. In this study we examined the expression of these tumor-associated CAs by immunohistochemistry in relation to necrosis and early breast tumor progression in 68 cases of ductal carcinoma in situ (DCIS) (39 pure DCIS and 29 DCIS associated with invasive carcinoma). CA IX expression was rare in normal epithelium and benign lesions, but was present focally in DCIS (50% of cases) and in associated invasive carcinomas (29%). In comparison, CA XII was frequently expressed in normal breast tissues (89%), in DCIS (84%), and in invasive breast lesions (71%). In DCIS, CA IX was associated with necrosis (P: = 0.0053) and high grade (P: = 0.012). In contrast, CA XII was associated with the absence of necrosis (P: = 0.036) and low grade (P: = 0.012). Despite this, augmented CA XII expression was occasionally observed adjacent to necrosis within high-grade lesions. Neither CA IX nor CA XII expression was associated with regional or overall proliferation as determined by MIB1 staining. Assessment of mammographic calcification showed that CA XII expression was associated with the absence of calcification (n = 43, P: = 0.0083). Our results demonstrate that induction of CA IX and CA XII occurs in regions adjacent to necrosis in DCIS. Furthermore, these data suggest that proliferation status does not influence expression of either CA in breast tissues, that hypoxia may be a dominant factor in the regulation of CA IX, and that factors related to differentiation, as determined by tumor grade, dominate the regulation of CA XII. The existence of differential regulation and associations with an aggressive phenotype may be important in the development of selective inhibitors of CAs, because the latter have recently been shown to prevent tumor invasion.

Hypoxia-Inducible Factor-1α Expression Predicts a Poor Response to Primary Chemoendocrine Therapy and Disease-Free Survival in Primary Human Breast Cancer

Purpose: To investigate the relationship of hypoxia-inducible factor-1α (HIF-1α) tumor expression in predicting the response to epirubicin and disease-free survival (DFS) in patients with breast cancer enrolled in a single institution trial of primary anthracycline and tamoxifen therapy. Experimental Design: The expression of HIF-1α was assessed by immunohistochemistry in 187 patients with T2-4 N0-1 breast cancer enrolled in a randomized trial comparing four cycles of single agent epirubicin versus epirubicin + tamoxifen as primary systemic treatment. All patients postoperatively received four cycles of the four weekly i.v. CMF regimen (cyclophosphamide, methotrexate, and 5-fluorouracil). Patients with estrogen receptor (ER)-positive primary tumors also underwent 5 years of treatment with adjuvant tamoxifen. Carbonic anhydrase IX (CAIX) was also scored as a marker of HIF activity. Results: Overall response to therapy progressively decreased with increasing tumor HIF-1α (P < 0.05), and HIF-1α was an independent predictor of response (P < 0.048). HIF-1α expression was also associated with a significantly shorter DFS (P < 0.02) in all patients and in ER-positive but not in ER-negative patients. Furthermore, CAIX positivity conferred a significantly shorter DFS (P = 0.02) compared with CAIX-negative tumors in patients with HIF-1α-negative tumors. Conclusions: HIF-1α expression in patients with breast cancer is a marker of poor therapy response and outcome, especially in ER-positive patients. The combination of two hypoxia markers has greater utility than assessing just one, and patients with hypoxia markers in their tumors may be suitable for administration of drugs that reduce HIF-1α expression and increase oxygen delivery to the tumor bed before starting neoadjuvant therapies.

The Androgen Receptor Is Significantly Associated with Vascular Endothelial Growth Factor and Hypoxia Sensing via Hypoxia-Inducible Factors HIF-1a, HIF-2a, and the Prolyl Hydroxylases in Human Prostate Cancer

Purpose: Hypoxia regulates key biological processes including angiogenesis via the transcription factor, hypoxia-inducible factor (HIF). In prostate cancer, angiogenesis is also influenced by androgens, and recent cell line studies suggest that this effect is partly mediated by HIF. The study aimed to assess whether a relationship exists in human prostate cancer between expression of the androgen receptor, HIFs, and the key angiogenesis factor, vascular endothelial growth factor (VEGF). Experimental Design: A tissue microarray comprised of 149 radical prostatectomy specimens was constructed. Semiquantitative immunohistochemical analysis was used to assess the expression of the androgen receptor, VEGF and HIF-1a and 2a, and their regulatory prolyl hydroxylase enzymes (PHD1, PHD2, and PHD3). Statistical analysis compared these factors with each other and with prostate-specific antigen relapse. Results: There was a significant correlation between HIF-1a and HIF-2a expression (P = 0.02), and with androgen receptor (P = 0.04 and P < 0.001, respectively) and VEGF expression (P = 0.05 and P < 0.001, respectively). VEGF was also significantly related to the androgen receptor (P = 0.05), whereas PHD2 was inversely related to HIF-2a expression. No significant association was shown between HIF-1a or HIF-2a and time to prostate-specific antigen recurrence (P = 0.20 and P = 0.94, respectively). Conclusions: These findings confirm the relationship between hypoxia and the androgen receptor in prostate cancer, and show for the first time, the role of HIF-2a in this disease process. They provide clinical evidence to support the recent cell line findings that androgens may regulate VEGF levels through the activation of HIF in androgen-sensitive tumors. Inhibition of both the HIF pathways may provide new therapeutic options in the management of this disease.

Phosphorylated ERα, HIF-1α, and MAPK Signaling As Predictors of Primary Endocrine Treatment Response and Resistance in Patients With Breast Cancer

PURPOSE We aimed to identify signaling pathways involved in the response and resistance to aromatase inhibitor therapy in patients with breast cancer. PATIENTS AND METHODS One hundred fourteen women with T2-4 N0-1, estrogen receptor (ER) alpha-positive tumors were randomly assigned to neoadjuvant letrozole or letrozole plus metronomic cyclophosphamide. Twenty-four tumor proteins involved in apoptosis, cell survival, hypoxia, angiogenesis, growth factor, and hormone signaling were assessed by immunohistochemistry in pretreatment samples (eg, caspase 3, phospho- mammalian target of rapamycin, hypoxia-inducible factor 1alpha [HIF-1alpha], vascular endothelial growth factor, mitogen-activated protein kinase [MAPK], phosphorylated epidermal growth factor receptor, phosphorylated ERalpha [pERalpha]). A multivariate generalized linear regression approach was applied using a penalized least-square minimization to perform variable selection and regularization. Ten-fold cross-validation and iterative leave-one-out were employed to validate and test the model, respectively. Tumor size, nodal status, age, tumor grade, histological type, and treatment were included in the analysis. RESULTS Ninety-one patients (81%) attained a disease response, 48 achieved a complete clinical response (43%) whereas 22 did not respond (19%). Increased pERalpha and decreased p44/42 MAPK were significant factors for complete response to treatment in all leave-one-out iterations. Increased p44/42 MAPK and HIF-1alpha were significant factors for treatment resistance in all leave-one-out iterations. There was no significant interaction between these variables and treatment. CONCLUSION Activated ERalpha form was an independent factor for sensitivity to chemoendocrine treatment, whereas HIF-1alpha and p44/42 MAPK were independent factors for resistance. Although further confirmatory analyses are needed, these findings have clear potential implications for future strategies in the management of clinical trials with aromatase inhibitors in the breast cancer.

The relation between hypoxia-inducible factor (HIF)-1α and HIF-2α expression with anemia and outcome in surgically treated head and neck cancer

Hypoxia promotes tumorigenesis through the hypoxia‐inducible factor (HIF) pathway. There are 2 main homologues of the regulatory proteins, HIF‐1α and HIF‐2α, which have different effects in genetic knock‐out experiments. Anemia may contribute to hypoxia by reducing oxygen delivery, but it is not known whether this influences HIF‐α expression in tumors.

The transcription factor DEC1 (stra13, SHARP2) is associated with the hypoxic response and high tumour grade in human breast cancers

DEC1, also known as SHARP-2 or Stra13, plays important roles in embryonic development, proliferation, apoptosis and cell differentiation in the mouse. DEC1 was recently identified as hypoxically induced in cDNA microarray studies of the human renal carcinoma cell line RCC4, to be regulated through hypoxia-inducible factor (HIF)-1α and via HIF-1α, able to block adipocyte differentiation. Nevertheless, its distribution and role in hypoxia and differentiation in human breast cancer are unknown. We therefore examined the pattern and level of expression of DEC1 using immunohistochemistry in whole tissue sections in normal, in situ and invasive breast carcinomas, and correlated the level of expression of DEC1 and clinicopathological factors and hypoxic tumour markers in 253 invasive carcinomas on tissue microarrays. We observed an increase in DEC1 expression during progression from normal to in situ and invasive carcinoma. Expression was not restricted to the tumour cell element but was also observed in endothelial, fibroblasts and inflammatory cells. There was a significant positive correlation between DEC1 and tumour grade (P=0.01), HIF-1α (P=0.04) and the hypoxically regulated gene angiogenin (P<0.0001), but no significant associations were observed with patient age (P=0.15), lymph node status (P=0.8), tumour size (P=0.3), oestrogen receptor (P=0.45), epidermal growth factor receptor (P=0.27) or Chalkley vessel count (P=0.45). There was no difference in relapse-free (P=0.84) or overall (P=0.78) survival. These findings suggest that DEC1 plays an important role in the progression to invasive breast cancer and that it may provide a mechanism by which hypoxia blocks tumour differentiation, and may contribute to a more aggressive phenotype. Reversing this phenotype may alter the biological behaviour of individual tumours.

Immunomodulation of FOXP3+ Regulatory T Cells by the Aromatase Inhibitor Letrozole in Breast Cancer Patients

Purpose: We have shown previously that tumor infiltration by FOXP3+ regulatory T cells (Treg) is associated with increased relapse and shorter survival of patients with both in situ and invasive breast cancer. Because estrogen regulates Treg numbers in mice and promotes the proliferation of human Tregs, we hypothesized that blocking estrogen receptor-α signaling would abrogate Tregs and be associated with response to hormonal therapy and increased survival. Experimental Design: FOXP3+ Tregs were quantified in tumor samples collected at baseline by incisional biopsy and after 6 months at definitive surgery in 83 elderly breast cancer patients (T2-4 N0-1) enrolled in a randomized phase II trial based on 6 months of primary letrozole (2.5 mg/d) or 6 months of letrozole plus oral “metronomic” cyclophosphamide (50 mg/d). Results: Treg number ranged from 0 to 380 (median, 30) before treatment and from 0 to 300 (median, 8) after treatment. There was a significant reduction in Tregs in letrozole and letrozole-cyclophosphamide patients (P < 0.0001 and P < 0.002, respectively) after treatment. Treg number at residual histology was inversely related with response (P < 0.03 and P = 0.50, respectively) and a greater Treg reduction was observed in responding patients (P < 0.03). Conclusion: This study suggests that aromatase inhibitors may have an indirect antitumor mechanism of action through reducing Tregs in breast tumors and may be of use in estrogen receptor-α-negative tumors in combination with immunotherapy approaches.

BNIP3 as a Progression Marker in Primary Human Breast Cancer; Opposing Functions in In situ Versus Invasive Cancer

Purpose: BNIP3 is involved in cell death and cell survival via autophagy. Its perinecrotic localization within ductal carcinoma in situ (DCIS) suggests an involvement in neoplastic cellular adaptation to low oxygen tension. This study has investigated the role of BNIP3 in normal and neoplastic breast. Experimental Design: Whole sections from 11 normal breast and microarrayed tissue cores from 81 DCIS and 251 invasive carcinomas were stained for BNIP3 and hypoxia-inducible factor-1α. The pattern and level of BNIP3 expression were correlated with clinicopathologic variables and hypoxia-inducible factor-1α. Results: BNIP3 expression was significantly up-regulated in the cytoplasm of DCIS and invasive carcinoma compared with normal breast (P = 0.0005 and P < 0.0001, respectively). Nuclear BNIP3 expression was associated with smaller tumor size (P = 0.04), low tumor grade (P = 0.005), and estrogen receptor positivity (P = 0.008) in invasive tumors. Nuclear BNIP3 expression was also associated with a longer disease-free survival among low-grade and estrogen receptor–positive tumors. (P = 0.03 and 0.04, respectively). Conversely, nuclear BNIP3 expression in DCIS was associated with a 3-fold increase in recurrence and a shorter disease-free survival (P = 0.03). Conclusions: Up-regulation of BNIP3 expression in DCIS and invasive carcinoma suggests a significant role in breast tumor progression. Its association with good survival outcome in invasive carcinoma but with an increased risk of recurrence and shorter disease-free survival in DCIS may suggest a pivotal switch from a cell death to survival function during the transition from preinvasive to invasive breast cancer.

CITED4 Inhibits Hypoxia-Activated Transcription in Cancer Cells, and Its Cytoplasmic Location in Breast Cancer Is Associated with Elevated Expression of Tumor Cell Hypoxia-Inducible Factor 1α

The interaction of hypoxia-inducible factor 1α and the CH1 domain of the transcriptional coactivator p300/CBP is necessary for the expression of hypoxia responsive genes and tumor angiogenesis. The transcription factor CITED2 binds p300/CBP at the CH1 domain and functions as a negative regulator of hypoxia signaling by competing with hypoxia-inducible factor 1α. CITED4, a recently identified member of the CITED family, binds p300/CBP via the CH1 domain and functions as a coactivator for transcription factor AP-2. Here, we show that CITED4 blocks the binding of hypoxia-inducible factor 1α to p300 in vitro and inhibits hypoxia-inducible factor-1α transactivation and hypoxia-mediated reporter gene activation. These studies suggest that CITED4 might function as an inhibitor of hypoxia-inducible factor 1α. To explore the function of CITED4 in breast cancer, we determined its expression in normal, in situ and invasive breast cancers. We also correlated its expression in 286 invasive breast tumors with clinicopathological, hypoxia markers and survival. In contrast to the nuclear localization of CITED4 in normal breast tissue, breast tumors were characterized by cytoplasmic and nuclear localization. Nuclear CITED4 expression was significantly inversely associated with tumor hypoxia-inducible factor 1α (P < 0.05), tumor size (P = 0.03), tumor grade (P = 0.0001), and Chalkley vessel count (P = 0.04). CITED4 showed no significant correlation with patient age (P = 0.45), estrogen receptor (P = 0.11), or epidermal growth factor receptor (P = 0.48). These results show that breast cancer development is characterized by either nuclear loss or cytoplasmic translocation of CITED4, with consequent loss of hypoxia-inducible factor-1α transcriptional antagonist activity. This may be an important mechanism by which tumors enhance hypoxia-inducible factor expression and result in an aggressive phenotype.

Down-Regulation of Phosphatidylinositol 3′-Kinase/AKT/Molecular Target of Rapamycin Metabolic Pathway by Primary Letrozole-Based Therapy in Human Breast Cancer

Purpose: The phosphatidylinositol 3′-kinase (PI3K)/AKT/molecular target of rapamycin (mTOR) pathway is involved in the development of tumor resistance to endocrine therapy in breast cancer cell lines and represents an attractive target for pharmacologic intervention. However, the effects of endocrine therapy with aromatase inhibitors on in vivo expression of this signaling cascade, and its relation to tumor response and patient outcome, is unknown. Experimental Design: PI3K, phospho-AKT (pAKT) and phospho-mTOR were assessed by immunohistochemistry on tumor specimens collected at baseline and after 6 months of treatment in 113 elderly breast cancer patients consecutively enrolled in a randomized phase II trial of primary letrozole therapy and letrozole associated with metronomic cyclophosphamide. Results: Basal expression of the pathway was not significantly correlated with response or patient outcome. Both letrozole alone and letrozole with cyclophosphamide resulted in a significant reduction of PI3K expression (P = 0.02 and P < 0.005, respectively) and phospho-mTOR expression (P = 0.0001 and P = 0.0001, respectively). pAKT showed no change in the letrozole arm, whereas it was significantly decreased in the letrozole plus cyclophosphamide arm (P < 0.005). pAKT expression reduction was associated with a greater response rate (P = 0.05) and greater reduction in Ki67 expression (P = 0.05). Phospho-mTOR expression reduction was associated with a significantly longer disease-free survival in a multivariate analysis (P = 0.02). Conclusions: Letrozole inhibits key molecules in the PI3K pathway that are important targets of new drugs being developed to overcome resistance. Changes in these molecules may have prognostic significance. These results should be taken into account when planning prospective trials testing up-front aromatase inhibitor with drugs targeting the PI3K/AKT/mTOR signaling pathway.