Paola Collecchi

The Stem Cell Factor–c-kit System and Mast Cells in Human Pancreatic Cancer

Stem cell factor (SCF) and its receptor c-kit take part in the regulation of developmental processes of mast cells, hematopoietic stem cells, and melanocytes, as well as in the growth control of human malignancies. To explore the possible role of the SCF–c-kit system and of mast cells in pancreatic cancer, the concomitant expression and distribution of the two molecules were examined in 17 normal and 26 cancerous human pancreatic tissues and in 6 cultured pancreatic cancer cell lines. Mast cell distribution was also evaluated in the same tissue samples. In addition, the effects of SCF and of the c-kit tyrosine-kinase inhibitor STI571 on the growth of the cancer cell lines and of the normal pancreatic ductal cell line TAKA-1 were assessed. SCF immunoreactivity was absent in acinar, ductal, and islet cells of the normal pancreas and faint in pancreatic cancer tissues and cell lines. In contrast, c-kit was clearly present in some normal and hyperplastic ducts of the normal pancreas, in the cancer cells of 73% of the tumor samples, and in all the cell lines tested. Mast cells, identified by tryptase and chymase immunostaining on consecutive tissue sections, showed immunoreactivity for SCF and c-kit in both normal and cancerous specimens and their number was significantly increased (p = 0.03) in pancreatic cancer compared with the normal pancreas. SCF showed a dose-dependent growth inhibitory effect on TAKA-1 cells (p < 0.001), whereas pancreatic cancer cells were resistant to the SCF-induced growth inhibition. Nonetheless, the growth of TAKA-1 cells and pancreatic cancer cells was inhibited by the c-kit tyrosine kinase inhibitor STI571. In conclusion, the SCF–c-kit system, possibly with the contribution of mast cells, may have a growth-regulating role in the normal pancreas, which is altered during malignant transformation.

Large needle aspiration biopsy and galectin-3 determination in selected thyroid nodules with indeterminate FNA-cytology.

Thyroid fine-needle aspiration biopsy (FNA)-cytology is widely used for the preoperative characterisation of thyroid nodules but this task is difficult for follicular lesions, which often remain undefined. We propose a strategy for improving the preoperative characterisation of selected follicular thyroid proliferations, which is based on large needle aspiration biopsy (LNAB) and galectin-3 expression analysis. Eighty-five thyroid specimens were obtained by LNAB (20-gauge needles) from thyroid nodules with indeterminate follicular FNA-cytology. Aspirated material was processed as a tissue microbiopsy to obtain cell blocks for both cyto/histo-morphological evaluation and galectin-3 expression analysis, by using a purified monoclonal antibody to galectin-3 and a biotin-free immunoperoxidase staining method. Preoperative diagnosis was compared to the final histology. LNAB and cell-block technique allow a preliminary distinction between nodules with a homogeneous microfollicular/trabecular structure, as frequently observed in tumours, and lesions with mixed normo–micro–macrofollicular architecture, as observed in goitre. Furthermore, LNAB provides optimal substrates for galectin-3 expression analysis. Among 85 cases tested, 14 galectin-3-positive cases were discovered preoperatively (11 thyroid cancers and three adenomas confirmed at the final histology), whereas galectin-3-negative cases were 71 (one carcinoma and 70 benign proliferations at the final histology). Sensitivity, specificity and diagnostic accuracy of this integrated morphologic and phenotypic diagnostic approach were 91.6, 97.2 and 95.3%, respectively. In conclusion, LNAB plus galectin-3 expression analysis when applied preoperatively to selected thyroid nodules candidate to surgery can potentially reduce unnecessary thyroid resections.

Fluvastatin synergistically enhances the antiproliferative effect of gemcitabine in human pancreatic cancer MIAPaCa-2 cells

The new combination between the nucleoside analogue gemcitabine and the cholesterol-lowering drug fluvastatin was investigated in vitro and in vivo on the human pancreatic tumour cell line MIAPaCa-2. The present study demonstrates that fluvastatin inhibits proliferation, induces apoptosis in pancreatic cancer cells harbouring a p21ras mutation at codon 12 and synergistically potentiates the cytotoxic effect of gemcitabine. The pharmacologic activities of fluvastatin are prevented by administration of mevalonic acid, suggesting that the shown inhibition of geranyl-geranylation and farnesylation of cellular proteins, including p21rhoA and p21ras, plays a major role in its anticancer effect. Fluvastatin treatment also indirectly inhibits the phosphorylation of p42ERK2/mitogen-activated protein kinase, the cellular effector of ras and other signal transduction peptides. Moreover, fluvastatin administration significantly increases the expression of the deoxycytidine kinase, the enzyme required for the activation of gemcitabine, and simultaneously reduces the 5′-nucleotidase, responsible for deactivation of gemcitabine, suggesting a possible additional role of these enzymes in the enhanced cytotoxic activity of gemcitabine. Finally, a significant in vivo antitumour effect on MIAPaCa-2 xenografts was observed with the simultaneous combination of fluvastatin and gemcitabine, resulting in an almost complete suppression and a marked delay in relapse of tumour growth. In conclusion, the combination of fluvastatin and gemcitabine is an effective cytotoxic, proapoptotic treatment in vitro and in vivo against MIAPaCa-2 cells by a mechanism of action mediated, at least in part, by the inhibition of p21ras and rhoA prenylation. The obtained experimental findings might constitute the basis for a novel translational research in humans.

THE SPECTRUM OF 67-kD LAMININ RECEPTOR EXPRESSION IN BREAST CARCINOMA PROGRESSION

Laminin is a glycoprotein of the basement membrane (BM), involved in a variety of normal and pathological cellular events including tumour invasion and metastasis. Cells bind laminin through different types of receptor. The 67‐kD laminin receptor (67LR) is a cell‐surface protein which binds laminin with high affinity. 67LR expression has been shown to increase in neoplastic cells, compared with normal tissues, and 67LR seems to play an important role during the first steps of neoplastic progression. In this study, 67LR expression was analysed during the morphological phases of breast cancer progression from normal tissue to invasive carcinoma. A total of 506 formalin‐fixed, paraffin‐embedded normal breast structures and lesions were stained by immunohistochemistry using the MLuC5 monoclonal antibody, which is specific for 67LR. The results show that in normal breast and in any kind of breast lesion, myoepithelial and endothelial cells express 67LR. While 67LR is not seen in the epithelium of normal breast, cysts, adenosis, and benign tumours, it is expressed in the epithelial cells of several hyperplasias and carcinomas in situ, both ductal and lobular, as well as in all invasive carcinomas. The 67LR‐positive cell subpopulation expands from hyperplastic lesions to invasive carcinoma, suggesting that 67LR could be related to the induction and progression of breast cancer.

Primary chemotherapy in locally advanced breast cancer (LABC): effects on tumour proliferative activity, bcl-2 expression and the relationship between tumour regression and biological markers

The rate of tumour cell proliferation evaluated by the [3H]-thymidine labelling index ([3H]-dT-LI) is known to be an independent prognostic factor in patients with operable breast cancer and significantly predicts the response to chemotherapy in patients with advanced disease. In locally advanced breast cancer (LABG), we examined whether chemotherapy induced modifications in [3H]-dt-LI, and bcl-2 expression and their relationship with tumour regression and prognosis. 70 LABC patients received three courses of primary chemotherapy (FEC: 5-fluorouracil 600 mg/m2, epidoxorubicin 60 mg/m2, cyclophosphamide 600 mg/m2, followed by surgery and subsequent adjuvant chemotherapy consisting of three courses of FEC alternated with three courses of CMF (cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, 5-fluorouracil 600 mg/m2). Tumour biological markers were evaluated on diagnostic biopsy, before primary chemotherapy and at surgery. Tumour cell proliferation was determined by [3H]-dT-LI, whilst bcl-2 expression was examined by immunohistochemical staining. The overall response rate to primary FEC was 74.3% (95% confidence interval 57.6-83.2%). The response rate correlated with high [3H]-dT-LI: 88% (29/33) of patients with high [3H]-dT-LI achieved an objective response compared with 62% (23/37) of patients with low [3H]-dT-LI (P = 0.014). The 3 patients achieving a pathological complete response after induction treatment had high proliferative tumours. The highest 2-year relapse free survival (66.6%) was observed in patients with low [3H]-dT-LI after primary chemotherapy. The median bcl-2 expression values before and after primary chemotherapy were 0% (range 0-80) and 30% (range 0-90), respectively (P = 0.03). Our data indicate that primary chemotherapy can modulate tumour cell kinetics and apoptosis-related genes. Pretreatment proliferative activity correlated with tumour response, whilst post-treatment [3H]-dT-LI correlated with relapse free survival.

Re-differentiation of thyroid carcinoma cell lines treated with 5-Aza-2′-deoxycytidine and retinoic acid.

We studied cell growth rate, mechanisms of growth inhibition, phenotype re-differentiation, expression of RARalpha, beta, gamma and differentiation thyroid genes before and after combined treatment with 5-Aza-CdR and RA (5-Aza/RA) of human thyroid carcinoma cell lines (FRO, WRO, TT). Furthermore, the activity and localization of the re-expressed sodium-iodide-symporter (NIS) protein was analyzed. After 5-Aza/RA treatment, all cell lines showed a significant reduction in cell growth. This was associated with apoptosis in the TT, with inhibition of cell proliferation in the WRO, and with cell cycle impairment in FRO and WRO. FRO and WRO treated with 5-Aza/RA lost the ability to grow in soft agar. FRO re-expressed thyroid transcription factor-1 and thyroglobulin, TT and WRO re-expressed PAX-8 and FRO and TT re-expressed RARbeta and NIS mRNA. Despite this expression, they were unable to take up iodine: a cytoplasmic localization of NIS protein was demonstrated in FRO. In conclusion, besides a significant reduction in cell growth rate and in the ability to grow in soft agar, treatment with 5-Aza/RA partially re-differentiated FRO and induced expression of NIS mRNA and protein in FRO and TT, but this treatment was unable to restore the functional activity of NIS, likely because it was located into the cytoplasm without reaching the plasma membrane.

Dysregulated PI3K/Akt/PTEN pathway is a marker of a short disease-free survival in node-negative breast carcinoma

The phosphoinositide 3-kinase/Akt pathway is involved in the pathogenesis of several human cancers. In this study, the biological and prognostic value of phosphoinositide 3-kinase/Akt pathway dysregulation was assessed by immunohistochemistry in a well-characterized series of 72 patients with node-negative breast cancer with a long-term follow-up. Phosphorylated Akt and PTEN expression was reduced in 32% and 12.5% of the tumors, respectively. Phosphorylated Akt or PTEN status was not associated with the main clinicopathologic and biological parameters, whereas their expression was tightly related to their downstream targets cyclin D1 and p27(Kip1) which are involved in cell proliferation. Survival analysis showed a strong association between a shorter disease-free survival and the dysregulated expression of phosphorylated Akt (P = .036), PTEN (P = .003), p27(Kip1) (P = .008), and Ki67 (P = .0007), or the distinct subtypes of breast tumors (luminal, HER2 overexpressing, and basal-like; P = .03). Moreover, multivariate analysis using the Cox proportional-hazards regression model showed that PTEN and Ki67 were independent predictive factors of disease recurrence and that their simultaneous dysregulation strongly increased the hazards ratio of the patients with node-negative breast cancer (hazards ratio, 38.30; P = .0014). In conclusion, our results show that the dysregulation of the phosphoinositide 3-kinase/Akt/PTEN pathway is relevant to the prognosis in node-negative breast carcinoma and that the evaluation of key components of this pathway might be a useful tool to identify the patients with node-negative breast cancer at high-risk of disease recurrence.

High level of messenger RNA for BRMS1 in primary breast carcinomas is associated with poor prognosis.

BRMS1 is regarded as a metastasis suppressor gene for its ability to reduce metastatic potential of human and murine breast cancer cells as well as human melanoma cells. However, BRMS1 association to human tumor progression is not clearly understood. In the present study we analyzed BRMS1 mRNA expression in tumor progression and its potential prognostic value for breast carcinoma. BRMS1 mRNA expression level was quantified by real‐time PCR in 47 tumoral, in 14 peritumoral and in 15 metastatic microdissected cellular populations from 47 breast cancer patients with 10‐year follow up. We found BRMS1 expression to be higher in carcinoma cells than in matching normal epithelial cell populations in 10 out of 14 cases (p = 0.0005), while lymph‐nodal carcinoma cells showed lower BRMS1 expression in 9 out of 15 cases (p = 0.001). Using both in vivo (human mammary breast carcinomas) and in vitro systems (breast cancer cell lines) we were able to demonstrate that BRMS1 overexpression was not a bias effect induced by cell proliferation rate. BRMS1 expression levels did not correlate with standard breast cancer prognostic factors but BRMS1 higher expression was associated with patient shorter disease‐free and overall survival. Our findings are apparently inconsistent with the concept of BRMS1 as a metastasis suppressor gene. One possible explanation is that epithelial cells increase their BRMS1 expression as a compensatory response to tumor formation or metastasis progression, which is elevated in proportion to tumor aggressiveness, whereas those cells of the primary tumor that cannot upregulate BRMS1 escape to form metastasis.

Nuclear Expression of Hypoxia-inducible Factor-1?? in Clear Cell Renal Cell Carcinoma is Involved in Tumor Progression

ObjectivesThe most frequent genomic abnormality in clear cell renal cell carcinoma (cc-RCC) is inactivation of Von Hippel-Lindau gene (VHL). pVHL19 is a ligase promoting proteosomal degradation of hypoxia-inducible factor-1alfa (HIF-1α); pVHL30 is associated with microtubules. VHL exert its oncogenetic action both directly and through HIF-1α activation. TNM classification is unable to define a correct prognostic evaluation of intracapsular cc-RCC. The nucleo-cytoplasmic trafficking in VHL/HIF-1α pathway could be relevant in understanding the molecular pathogenesis of renal carcinogenesis. This study analyzes VHL/HIF-1α proteins in a large series of intracapsular cc-RCCs, correlating their expression and cellular localization with prognosis. Materials and MethodsTwo anti-pVHL (clones Ig32 and Ig33) and 1 anti-HIF-1α were used on tissue microarrays from 136 intracapsular cc-RCCs (mean follow-up: 74 mo). Clone 32 recognizes both pVHLs, whereas clone 33 only pVHL30. Results were matched with clinicopathologic variables and tumor-specific survival (TSS). ResultsA strong cytoplasmic positivity was found for all antibodies in the largest part of cases, associated to a strong nuclear localization in the case of HIF-1α. All pVHL-negative cases were associated with high HIF-1α expression. pVHL negativity and HIF-1α nuclear positivity significantly correlated with shorter TSS. In multivariate analysis both pVHL negativity and HIF-1α nuclear expression were independent predictors of TSS. ConclusionsThe localization of the proteins well matches with their role and with the supposed tumor molecular pathways. The correlation with prognosis of VHL/HIF-1α alterations confirms the relevance of their molecular pathway and of the cellular trafficking of their products in the pathogenesis of renal cancer.

Cyclins of phases G1, S and G2/M are overexpressed in aneuploid mammary carcinomas.

Expression of cyclins A, B1 and D1 in human breast cancer was analyzed using dual-parameter flow cytometry with simultaneous evaluation of the DNA content. The asynchronous MCF-7 breast adenocarcinoma cells were used to implement flow cytometry analysis and to analyze the cell cycle distribution of cyclins. The patterns of the cyclin expression were also analyzed in vivo in fresh tissue specimens of human breast carcinomas. The combined measurement of DNA and cyclins showed a higher cyclin expression in aneuploid (11.5 +/- 2.0%, 4.3 +/- 1.1%, and 19.5 +/- 3.4% positive cells for cyclins A, B, and D1, respectively) than in diploid carcinomas (3.9 +/- 1.2%, 1.1 +/- 0.4%, and 5.0 +/- 1.2% positive cells for cyclins A, B, and D1, respectively). A positive relationship was also found between cyclin A and D1 expression and H(3)-thymidine labeling index. In the in vitro model, the asynchronous growing MCF-7 cells showed a variable number of cells expressing cyclins in an unscheduled way, unrelated to the phase at which these cyclins are expressed in normal cells. A similar condition was also observed in tumors. In conclusion, the data showed a deregulated expression of cyclins in a transformed adenocarcinoma cell line and in breast tumors. Furthermore, overexpression of these proteins is related to the aneuploid and high proliferative activity of human mammary carcinomas.