Giulietta A. Perrone

Modulators of HIF1α and NFkB in Cancer Treatment: Is it a Rational Approach for Controlling Malignant Progression?

HIF1α and NFkB are two transcription factors very frequently activated in tumors and involved in tumor growth, progression, and resistance to chemotherapy. In fact, HIF1α and NFkB together regulate transcription of over a thousand genes that, in turn, control vital cellular processes such as adaptation to the hypoxia, metabolic reprograming, inflammatory reparative response, extracellular matrix digestion, migration and invasion, adhesion, etc. Because of this wide involvement they could control in an integrated manner the origin of the malignant phenotype. Interestingly, hypoxia and inflammation have been sequentially bridged in tumors by the discovery that alarmin receptors genes such as RAGE, P2X7, and some TLRs, are activated by HIF1α; and that, in turn, alarmin receptors strongly activate NFkB and proinflammatory gene expression, evidencing all the hallmarks of the malignant phenotype. Recently, a large number of drugs have been identified that inhibit one or both transcription factors with promising results in terms of controlling tumor progression. In addition, many of these molecules are natural compounds or off-label drugs already used to cure other pathologies. Some of them are undergoing clinical trials and soon they will be used alone or in combination with standard anti-tumoral agents to achieve a better treatment of tumors with reduction of metastasis formation and, more importantly, with a net increase in survival. This review highlights the central role of HIF1α activated in hypoxic regions of the tumor, of NFkB activation and proinflammatory gene expression in transformed cells to understand their progression toward malignancy. Different molecules and strategies to inhibit these transcription factors will be reviewed. Finally, the central role of a new class of deacetylases called Sirtuins in regulating HIF1α and NFkB activity will be outlined.

Overexpression of estrogen receptor-α in human papillary thyroid carcinomas studied by laser- capture microdissection and molecular biology.

The expression pattern of estrogen receptor (ER) isoforms in normal and tumor thyroid tissues is still controversial and poor defined, therefore, a more detailed study of the distribution of these molecules is needed. Most discrepancies might be due to the methods utilized. We studied the expression of ER isoforms in human papillary thyroid carcinoma (PTC), in fine‐needle aspiration biopsy‐derived specimens, and in cells, using more accurate techniques, such as laser‐capture microdissection, real‐time quantitative PCR, and Western blot. Laser‐capture microdissection allowed us to isolate homogeneous cell populations from human PTC surgical samples. Tumor, peritumor, or normal host tissue of the same sample were separately dissected and analyzed by RT‐PCR and Western blot. Estrogen receptor‐α mRNA was more expressed in cancer‐microdissected cells from human PTC, as compared with microdissected cells obtained from surrounding normal host tissue (450 vs 12, P = 0.001). A similar pattern was observed with Western blot for the ER‐α protein. By contrast, ER‐β mRNA expression was not detected among the microdissected tissue fractions. Fine‐needle aspiration biopsy‐derived specimens showed a similar expression pattern to ER. Moreover, human PTC cell line BCPAP and cancer stem cells from PTC, analyzed under hypoxic conditions, showed a hypoxia‐driven increase in ER‐α expression. In conclusion, ER‐α might have an important role in human PTC, and its overexpression can be studied in routine needle aspirate as a possible marker of malignancy. (Cancer Sci 2011; 102: 1921–1927)

Exosomal clusterin, identified in the pericardial fluid, improves myocardial performance following MI through epicardial activation, enhanced arteriogenesis and reduced apoptosis.

BACKGROUND We recently demonstrated that epicardial progenitor cells participate in the regenerative response to myocardial infarction (MI) and factors released in the pericardial fluid (PF) may play a key role in this process. Exosomes are secreted nanovesicles of endocytic origin, identified in most body fluids, which may contain molecules able to modulate a variety of cell functions. Here, we investigated whether exosomes are present in the PF and their potential role in cardiac repair. METHODS AND RESULTS Early gene expression studies in 3day-infarcted mouse hearts showed that PF induces epithelial-to-mesenchymal transition (EMT) in epicardial cells. Exosomes were identified in PFs from non-infarcted patients (PFC) and patients with acute MI (PFMI). A shotgun proteomics analysis identified clusterin in exosomes isolated from PFMI but not from PFC. Notably, clusterin has a protective effect on cardiomyocytes after acute MI in vivo and is an important mediator of TGFβ-induced. Clusterin addition to the pericardial sac determined an increase in epicardial cells expressing the EMT marker α-SMA and, interestingly, an increase in the number of epicardial cells ckit(+)/α-SMA(+), 7days following MI. Importantly, clusterin treatment enhanced arteriolar length density and lowered apoptotic rates in the peri-infarct area. Hemodynamic studies demonstrated an improvement in cardiac function in clusterin-treated compared to untreated infarcted hearts. CONCLUSIONS Exosomes are present and detectable in the PFs. Clusterin was identified in PFMI-exosomes and might account for an improvement in myocardial performance following MI through a framework including EMT-mediated epicardial activation, arteriogenesis and reduced cardiomyocyte apoptosis.

Bridging hypoxia, inflammation and estrogen receptors in thyroid cancer progression

Thyroid cancer is a common endocrine-related cancer with a higher incidence in women than in men. Thyroid tumors are classified on the basis of their histopathology as papillary, follicular, medullary, and undifferentiated or anaplastic. Epidemiological and in vitro or in vivo studies have suggested a correlation between incidence of thyroid malignancies and hormones. In particular, growing evidence indicates a role of estrogens and estrogen receptors (ERs) in thyroid tumorigenesis, reprogramming and progression. In this scenario, estrogens are hypothesized to contribute to the observed female predominance of thyroid cancer in reproductive years. However, the precise contribution of estrogens in thyroid proliferative disease initiation and progression is not well understood. HIF-1α and NF-κB are two transcription factors very frequently activated in tumors and involved in tumor growth, progression and resistance to chemotherapy. In fact, HIF-1α and NF-κB together regulate transcription of over a thousand genes that, in turn, control vital cellular processes such as adaptation to the hypoxia, metabolic and differentiation reprogramming, inflammatory-reparative response, extracellular matrix digestion, migration and invasion, adhesion, etc. Because of this wide involvement, they could control in an integrated manner the origin of the malignant phenotype. Interestingly, hypoxia and inflammation have been sequentially bridged in tumors by the discovery that alarmin receptors genes such as RAGE, P2X7 and some TLRs are activated by HIF-1α; and that, in turn, alarmin receptors strongly activate NF-κB and proinflammatory gene expression, evidencing all the hallmarks of the malignant phenotype. Recently, a large number of drugs have been identified that inhibit one or both transcription factors with promising results in terms of controlling tumor progression. In addition, many of these inhibitors are natural compounds or off-label drugs already used to cure other pathologies. Some of them are undergoing clinical trials and soon they will be used alone or in combination with standard anti-tumoral agents to achieve a better treatment of tumors to achieve a reduction of metastasis formation and, more importantly, a net increase in survival. This review highlights the central role of HIF-1α activated in hypoxic regions of the tumor, of NF-κB activation and proinflammatory gene expression in transformed thyroid cells to understand their progression toward malignancy. The role of ER-α will be underlined, considering also its role in reprogramming cancer cells.

Acute Myopathy Associated with Chronic Licorice Ingestion: Reversible Loss of Myoadenylate Deaminase Activity

A patient with acute rhabdomyolysis and absence of myoadenylate deaminase (MADA) associated with chronic licorice intoxication is presented. Clinical and laboratory examination of the patient and morphologic study over skeletal muscle were performed. The major effect of licorice intoxication is hypokalemia, which may explain most of the observed clinical symptoms and morphological changes. The absence of MADA may be a consequence of the direct toxic effect of licorice glycosides. To our knowledge, this is the first report in which a lack of MADA and chronic licorice intoxication has been shown to be associated with clinical, histochemical, biochemical, and morphological changes, which were completely reversed with potassium supplementation and licorice withdrawal.

Overexpression of estrogen receptor alpha in human thyroid papillary carcinoma (TPC)

TPC is 3-4 fold more common in premenopausal women than in men, suggesting an involvement of estrogens and their receptors (ERs) in the growth and progression of this tumor. ER alpha (ER-A) and ER beta (ER-B) play a different role in the natural history of tumors originating from breast, endometrium and prostate epithelia, the target-tissues of estrogens, ER-A having a proliferative and antiapoptotic activity, while ER-B displaying differentiative and proapoptotic effects. The expression of ER isoforms in normal and TPC tissues is still controversial, therefore a more detailed study of distribution of these molecules is needed. Immunohistochemistry appears to be inadequate for reliable quantitative studies. In the molecular biology approach, nucleic acid and protein extraction from whole tissue have generated highly variable data due to the confounding inconstant cellular populations of tissue samples. In our study we have used the laser capture microdissection to obtain homogeneous leukocyte-free tissue fractions from surgical samples, dissecting only epithelial tissue from tumor or normal host tissue of the same sample. Then they have been analyzed by HT real-time PCR and by WB. Our results demonstrate that ER-A is constantly overexpressed (200-300 folds) in epithelia microdissected from TPC, as compared with fractions obtained from surrounding normal host tissue. A similar pattern was observed with WB for the ER-A protein. For ER-B we were not able to evidence any change in expression or in amount of protein among different tissue fractions. To explore a possible clinical use of these data, we have studied 32 needle-aspirate from TPC and 3 from benign goiter lesions, evidencing a similar pattern of ER isoforms, as observed in microdissected tissues. In conclusion, ER-A may have an important role in the natural history of human TPC already evidenced for classical estrogen target tissues and its overexpression may be evidenced also in routine needle aspirate.