Adjuvant therapy with GnRH agonists/tamoxifen in breast cancer should be a good council for patients with hormone receptor-positive tumours and wish to preserve fertility

Infertility represents one of the main long-term consequences of the chemotherapy used for the adjuvant treatment of breast cancer. Approximately 60-65% of breast cancers express the nuclear hormone receptor in premenopausal women. Adjuvant endocrine therapy is an integral component of care for patients with hormone receptor-positive (HR+) tumours. The GnRH agonist (GnRHa) alone or in combination with tamoxifen produces results at least similar to those obtained with the different chemotherapy protocols in patients with HR+ breast cancer with respect to recurrence-free survival and overall survival. It is time to indicate adjuvant therapy with GnRHa associated with tamoxifen for patients with breast cancer (HR+ tumours) if they want to preserve their reproductive function. The evaluation of ovarian reserve tests: follicle stimulating hormone (FSH), anti-Mullerian hormone (AMH), inhibin B, antral follicle count (AFC) and ovarian volume 6 months, and 1 year after the end of therapy with GnRHa/tamoxifen must be realised. The recurrence-free survival and overall survival should be analysed. The major implication of this hypothesis will be to avoid adjuvant chemotherapy for patients with breast cancer (HR+ tumours) that request fertility preservation. It is expected that ovarian function should not be altered in almost all cases and subsequent pregnancy a real possibility. (C) 2012 Elsevier Ltd. All rights reserved.

Adjuvant therapy with GnRH agonists/tamoxifen in breast cancer should be a good council for patients with hormone receptor-positive tumours and wish to preserve fertility

Infertility represents one of the main long-term consequences of combination chemotherapy used for the treatment of breast cancer. Approximately 60%-65% of breast cancers express the nuclear hormone receptor in premenopausal women. Adjuvant endocrine therapy is an integral component of care for patients with hormone receptor-positive (HR+) tumours. The GnRH agonist (GnRHa) alone or in combination with tamoxifen produces results at least similar to those obtained with the different chemotherapy protocols in patients with HR+ tumors with respect to recurrence-free survival and overall survival, Presentation of the hypothesis: It is time to indicate adjuvant therapy with GnRHa associated with tamoxifen for patients with breast cancer (HR+ tumours) if they want to preserve their reproductive function. Testing the hypothesis: Assessment of ovarian reserve tests: follicle stimulating hormone (FSH), anti-Mullerian hormone (AMH), inhibin B, antral follicle count (AFC) and ovarian volume 6 months, and 1 year after the end of therapy with GnRHa/tamoxifen. The recurrence-free survival and overall survival should be analysed. Implications of the hypothesis: The major implication will be to avoid adjuvant chemotherapy for patients with breast cancer (HR+ tumours) that request fertility preservation. It is expected that ovarian function should not be altered in almost all cases. © Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida.

Prognostic significance of CD8+ T lymphocytes in breast cancer depends upon both oestrogen receptor status and histological grade

Results of previous studies on the influence of tumour infiltrating lymphocytes on prognosis of women with breast cancer have been mixed. This study re-evaluates the role of tumour-infiltrating lymphocytes as a prognostic marker in women with breast cancer.

Human epidermal growth factor receptor-2 gene amplification in gastric cancer using tissue microarray technology

To assess human epidermal growth factor receptor-2 (HER2)-status in gastric cancer and matched lymph node metastases by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH).

Targeting the insulin-like growth factor-1 receptor in human cancer

The insulin-like growth factor (IGF) signaling system plays a crucial role in human cancer and the IGF-1 receptor (IGF-1R) is an attractive drug target against which a variety of novel anti-tumor agents are being developed. Deregulation of the IGF signaling pathway frequently occurs in human cancer and involves the establishment of autocrine loops comprising IGF-1 or IGF-2 and/or IGF-1R over-expression. Epidemiologic studies have documented a link between elevated IGF levels and the development of solid tumors, such as breast, colon, and prostate cancer. Anti-cancer strategies targeting the IGF signaling system involve two main approaches, namely neutralizing antibodies and small molecule inhibitors of the IGF-1R kinase activity. There are numerous reports describing anti-tumor activity of these agents in pre-clinical models of major human cancers. In addition, multiple clinical trials have started to evaluate the safety and efficacy of selected IGF-1R inhibitors, in combination with standard chemotherapeutic regimens or other targeted agents in cancer patients. In this mini review, I will discuss the role of the IGF signaling system in human cancer and the main strategies which have been so far evaluated to target the IGF-1R.

Cell Cycle Regulatory Roles of Estrogen Receptor alpha (ERα) in Breast Cancer Cells

Previous studies have shown that Estrogen Receptor alpha (ERα) is an important indicator for diagnosis, prognosis and treatment of breast cancers. However, the question remains as to the role of ERα in the cell in the presence versus absence of 17-β estradiol In this dissertation the role of ERα in both its unliganded and liganded state, with respect to the cell cycle will be explored. The cell line models used in this project are ER-positive MCF-7 cells with and without siRNA to ERα and ER-positive MDA-MB-231 cells that have been engineered to express ERα. Cells were synchronized and the cell cycle progression was monitored by flow cytometric analysis. Using these methods, two specific questions were addressed: Does ERα modulate the cell cycle differently under liganded versus unliganded conditions? And, does the presence of ERα regulate cell cycle phase transitions? The results show for the first time that ERα is cell cycle regulated and modulates the progression of cells through S and G2/M phases of the cell cycle. Ligand bound ERα increases progression through S and G2/M phases, whereas unliganded ERα acts as an inhibitor of cell cycle progression. To further investigate the cell cycle regulated effects of liganded ERα, a luciferase assay was performed and showed that the transcription of target genes such as Progestrone Receptor (PgR) and Trefoil protein (pS2) increased duing S and G2/M phases when ERα is bound to ligand. Additionally, complex formation between cyclin B and ER α was shown by immunoprecipitation and led to the discovery that anaphase promoting complex (APC) is the E3 ligase for both cyclin B and ERα at the termination of M phase. Our findings suggest that unliganded ERα has an inhibitory effect on the progression of the cell cycle. Therefore, it is reasonable to speculate that the combination of drugs that lower estrogen level (such as aromatase inhibitors) and preserves ERα from degradation would provide better outcome for breast cancer treatment. We have shown that APC functions as the E3 ligase for ERα and thus might provide a target to design a specific inhibitor of ERα degradation.

APPLICATIONS OF EPHB4 RECEPTOR SPECIFIC PEPTIDES IN TARGETED CANCER IMAGING AND THERAPY

EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are found over-expressed in a variety of tumors cells including glioma cells as well as angiogenic blood vessels. Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed against EphB4, and improve patient outcomes. Targeted delivery of EphB4 receptor specific peptide conjugated hollow gold nanoshells (HAuNS) into tumors has great potential in cancer imaging and photothermal therapy. In this study, we developed an EphB4 specific peptide named TNYL-RAW and labeled with radioisotope 64Cu and Cy5.5 dye. We also conjugate this specific peptide with hollow gold nanoshells (HAuNS) to evaluate targeted photothermal therapy of cancers. In vitro, 64Cu-DOTA-TNYL- RAW specifically bind to CT26 and PC-3M cells but not to A549 cells. In vivo, Small-animal PET/CT clearly showed the significant uptake of 64Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors. Furthermore, µPET/CT and near-infrared optical imaging clearly showed the uptake of the dual labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of nude mice. In U251 tumors, Cy5.5-labeled peptide can bind to EphB4-expressing tumor blood vessels and tumors cells. But in U87 models, dual labeled peptide only could bind to tumor associated blood vessels. Also, Irradiation of PC-3M and CT-26 cell treated with TNYL-PEG-HAuNS nanopatilces with near-infrared (NIR) laser resulted in selective destruction of these cells in vitro. EphB4 targeted TNYL-PEG-HAuNS showed more photothermal killing effect on CT26 tumor model than PEG-HAuNS did. In summary, tumors with overexpression of EphB4 receptors can be noninvasively visualized by micro PET/CT with 64Cu labeled or dual labeled TNYL-RAW peptide. Targeted delivery of TNYL-RAW conjugated HAuNS into tumors can greatly improve the treatment effect of photothermal therapy. The information acquired with this study should be advantageous in improving diagnostics and future applications in photothermal ablation therapy in clinical.

Myoglobin expression in prostate cancer is correlated to androgen receptor expression and markers of tumor hypoxia.

Recent studies identified unexpected expression and transcriptional complexity of the hemoprotein myoglobin (MB) in human breast cancer but its role in prostate cancer is still unclear. Expression of MB was immunohistochemically analyzed in three independent cohorts of radical prostatectomy specimens (n = 409, n = 625, and n = 237). MB expression data were correlated with clinicopathological parameters and molecular parameters of androgen and hypoxia signaling. Expression levels of novel tumor-associated MB transcript variants and the VEGF gene as a hypoxia marker were analyzed using qRT-PCR. Fifty-three percent of the prostate cancer cases were MB positive and significantly correlated with androgen receptor (AR) expression (p < 0.001). The positive correlation with CAIX (p < 0.001) and FASN (p = 0.008) as well as the paralleled increased expression of the tumor-associated MB transcript variants and VEGF suggest that hypoxia participates in MB expression regulation. Analogous to breast cancer, MB expression in prostate cancer is associated with steroid hormone signaling and markers of hypoxia. Further studies must elucidate the novel functional roles of MB in human carcinomas, which probably extend beyond its classic intramuscular function in oxygen storage.

18F-RB390: innovative ligand for imaging the T877A androgen receptor mutant in prostate cancer via positron emission tomography (PET).

BACKGROUND Detecting prostate cancer before spreading or predicting a favorable therapy are challenging issues for impacting patient's survival. Presently, 2-[(18) F]-fluoro-2-deoxy-D-glucose ((18) F-FDG) and/or (18) F-fluorocholine ((18) F-FCH) are the generally used PET-tracers in oncology yet do not emphasize the T877A androgen receptor (AR) mutation being exclusively present in cancerous tissue and escaping androgen deprivation treatment. METHODS We designed and synthesized fluorinated 5α-dihydrotestosterone (DHT) derivatives to target T877A-AR. We performed binding assays to select suitable candidates using COS-7 cells transfected with wild-type or T877A AR (WT-AR, T877A-AR) expressing plasmids and investigated cellular uptake of candidate (18) F-RB390. Stability, biodistribution analyses and PET-Imaging were assessed by injecting (18) F-RB390 (10MBq), with and without co-injection of an excess of unlabeled DHT in C4-2 and PC-3 tumor bearing male SCID mice (n = 12). RESULTS RB390 presented a higher relative binding affinity (RBA) (28.1%, IC50  = 32 nM) for T877A-AR than for WT-AR (1.7%, IC50  = 357 nM) related to DHT (RBA = 100%). A small fraction of (18) F-RB390 was metabolized when incubated with murine liver homogenate or human blood for 3 hr. The metabolite of RB390, 3-hydroxysteroid RB448, presented similar binding characteristics as RB390. (18) F-RB390 but not (18) F-FDG or (18) F-FCH accumulated 2.5× more in COS-7 cells transfected with pSG5AR-T877A than with control plasmid. Accumulation was reduced with an excess of DHT. PET/CT imaging and biodistribution studies revealed a significantly higher uptake of (18) F-RB390 in T877A mutation positive xenografts compared to PC-3 control tumors. This effect was blunted with DHT. CONCLUSION Given the differential binding capacity and the favorable radioactivity pattern, (18) F-RB390 represents the portrayal of the first imaging ligand with predictive potential for mutant T877A-AR in prostate cancer for guiding therapy. Prostate 75:348-359, 2015. © 2014 Wiley Periodicals, Inc.

Regulation of estrogen receptor alpha by the tumor suppressor p53 in breast cancer cells

Estrogen receptor (ER) and the tumor suppressor p53 are key prognostic indicators in breast cancer. Estrogen signaling through its receptor (ER) controls proliferation of normal as well as transformed mammary epithelial cells, and the presence of ER is established as a marker of good prognosis and response to therapy. The p53 tumor suppressor gene is often referred to as the "cellular gatekeeper" due to its extensive control of cell proliferation and apoptosis. Loss of functional p53 is a negative prognostic indicator and is correlated with lack of response to antiestrogens, reduced disease-free interval and increased chance of disease recurrence. Clinical studies have demonstrated that tumors with mutated p53 tend to be ER negative, while ER positive tumors tend to have wild type p53. ^ Recent studies from our lab indicate that p53 genotype correlates with estrogen receptor expression in mammary tumors in vivo. We therefore hypothesized that p53 regulates ER expression in mammary cancer cells by recruitment of specific cofactors to the ER promoter. To test this, MCF-7 cells were treated with doxorubicin or ionizing radiation, both of which stimulated significant increases in p53 expression, as expected, but also increased ER expression in a p53-dependent manner. Furthermore, in cells treated with siRNA targeting p53, both p53 and ER protein levels were significantly reduced. P53 was also demonstrated to transcriptionally regulate the ER promoter in luciferase assays and chromatin immunoprecipitation assays showed that p53 was recruited to the ER promoter along with CARM1, CBP, c-Jun and Sp1 and that this multifactor complex was formed in a p53-dependent manner. The regulation of ER by p53 has therapeutic implications, as the treatment of breast cancer cells with doxorubicin sensitized these cells to tamoxifen treatment. Furthermore, response to tamoxifen as well as to estrogen was dependent on p53 expression in ER positive human breast cancer cells. Taken together, these data demonstrate that p53 regulates ER expression through transcriptional control of the ER promoter, accounting for their concordant expression in human breast cancer and identifying potentially beneficial therapeutic strategies for the treatment of ER positive breast cancers. ^

The Role of Type I Insulin-Like Growth Factor Receptor Signaling in Breast Cancer Brain Metastasis

Brain metastasis is a common cause of mortality in cancer patients. Approximately 20-30% of breast cancer patients acquire brain metastasis, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF- IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that the IGF-IR signaling axis is constitutively active in brain-seeking sublines of breast cancer cells, driving an increase in in vitro metastatic properties. We demonstrate that IGF-IR signaling is activated in an autocrine manner as a result of IGFBP3 overexpression in brain-seeking cells. Transient and stable knockdown of IGF-IR results in a downregulation of IGF-IR downstream signaling through phospho-AKT, as well as decreased in vitro migration and invasion of MDA- MB-231Br brain-seeking cells. Using an in vivo experimental brain metastasis model, we show that IGF-IR ablation attenuates the establishment of brain metastases and prolongs survival. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.

Activating mutations for the Met tyrosine kinase receptor in human cancer

Recently, mutations in the Met tyrosine kinase receptor have been identified in both hereditary and sporadic forms of papillary renal carcinoma. We have introduced the corresponding mutations into the met cDNA and examined the effect of each mutation in biochemical and biological assays. We find that the Met mutants exhibit increased levels of tyrosine phosphorylation and enhanced kinase activity toward an exogenous substrate when compared with wild-type Met. Moreover, NIH 3T3 cells expressing mutant Met molecules form foci in vitro and are tumorigenic in nude mice. Enzymatic and biological differences were evident among the various mutants examined, and the somatic mutations were generally more active than those of germ-line origin. A strong correlation between the enzymatic and biological activity of the mutants was observed, indicating that tumorigenesis by Met is quantitatively related to its level of activation. These results demonstrate that the Met mutants originally identified in human papillary renal carcinoma are oncogenic and thus are likely to play a determinant role in this disease, and these results raise the possibility that activating Met mutations also may contribute to other human malignancies.

Prostacyclin-mediated activation of peroxisome proliferator-activated receptor δ in colorectal cancer

There is evidence from both genetic and pharmacologic studies to suggest that the cyclooxygenase-2 (COX-2) enzyme plays a causal role in the development of colorectal cancer. However, little is known about the identity or role of the eicosanoid receptor pathways activated by COX-derived prostaglandins (PG). We previously have reported that COX-2-derived prostacyclin promotes embryo implantation in the mouse uterus via activation of the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) δ. In light of the recent finding that PPARδ is a target of β-catenin transactivation, it is important to determine whether this signaling pathway is operative during the development of colorectal cancer. Analysis of PPARδ mRNA in matched normal and tumor samples revealed that expression of PPARδ, similar to COX-2, is up-regulated in colorectal carcinomas. In situ hybridization studies demonstrate that PPARδ is expressed in normal colon and localized to the epithelial cells at the very tips of the mucosal glands. In contrast, expression of PPARδ mRNA in colorectal tumors was more widespread with increased levels in transformed epithelial cells. Analysis of PPARδ and COX-2 mRNA in serial sections suggested they were colocalized to the same region within a tumor. Finally, transient transfection assays established that endogenously synthesized prostacyclin (PGI2) could serve as a ligand for PPARδ. In addition, the stable PGI2 analog, carbaprostacyclin, and a synthetic PPARδ agonist induced transactivation of endogenous PPARδ in human colon carcinoma cells. We conclude from these observations that PPARδ, similar to COX-2, is aberrantly expressed in colorectal tumors and that endogenous PPARδ is transcriptionally responsive to PGI2. However, the functional consequence of PPARδ activation in colon carcinogenesis still needs to be determined.

Conversion of a radioresistant phenotype to a more sensitive one by disabling erbB receptor signaling in human cancer cells

Inhibition of cell growth and transformation can be achieved in transformed glial cells by disabling erbB receptor signaling. However, recent evidence indicates that the induction of apoptosis may underlie successful therapy of human cancers. In these studies, we examined whether disabling oncoproteins of the erbB receptor family would sensitize transformed human glial cells to the induction of genomic damage by γ-irradiation. Radioresistant human glioblastoma cells in which erbB receptor signaling was inhibited exhibited increased growth arrest and apoptosis in response to DNA damage. Apoptosis was observed after radiation in human glioma cells containing either a wild-type or mutated p53 gene product and suggested that both p53-dependent and -independent mechanisms may be responsible for the more radiosensitive phenotype. Because cells exhibiting increased radiation-induced apoptosis were also capable of growth arrest in serum-deprived conditions and in response to DNA damage, apoptotic cell death was not induced simply as a result of impaired growth arrest pathways. Notably, inhibition of erbB signaling was a more potent stimulus for the induction of apoptosis than prolonged serum deprivation. Proximal receptor interactions between erbB receptor members thus influence cell cycle checkpoint pathways activated in response to DNA damage. Disabling erbB receptors may improve the response to γ-irradiation and other cytotoxic therapies, and this approach suggests that present anticancer strategies could be optimized.

From transforming growth factor-β signaling to androgen action: Identification of Smad3 as an androgen receptor coregulator in prostate cancer cells

Although transforming growth factor-β (TGF-β) has been identified to mainly inhibit cell growth, the correlation of elevated TGF-β with increasing serum prostate-specific antigen (PSA) levels in metastatic stages of prostate cancer has also been well documented. The molecular mechanism for these two contrasting effects of TGF-β, however, remains unclear. Here we report that Smad3, a downstream mediator of the TGF-β signaling pathway, functions as a coregulator to enhance androgen receptor (AR)-mediated transactivation. Compared with the wild-type AR, Smad3 acts as a strong coregulator in the presence of 1 nM 5α-dihydrotestosterone, 10 nM 17β-estradiol, or 1 μM hydroxyflutamide for the LNCaP mutant AR (mtAR T877A), found in many prostate tumor patients. We further showed that endogenous PSA expression in LNCaP cells can be induced by 5α-dihydrotestosterone, and the addition of the Smad3 further induces PSA expression. Together, our findings establish Smad3 as an important coregulator for the androgen-signaling pathway and provide a possible explanation for the positive role of TGF-β in androgen-promoted prostate cancer growth.