Paola Cassoni

Stromal contribution to the colorectal cancer transcriptome

Recent studies identified a poor-prognosis stem/serrated/mesenchymal (SSM) transcriptional subtype of colorectal cancer (CRC). We noted that genes upregulated in this subtype are also prominently expressed by stromal cells, suggesting that SSM transcripts could derive from stromal rather than epithelial cancer cells. To test this hypothesis, we analyzed CRC expression data from patient-derived xenografts, where mouse stroma supports human cancer cells. Species-specific expression analysis showed that the mRNA levels of SSM genes were mostly due to stromal expression. Transcriptional signatures built to specifically report the abundance of cancer-associated fibroblasts (CAFs), leukocytes or endothelial cells all had significantly higher expression in human CRC samples of the SSM subtype. High expression of the CAF signature was associated with poor prognosis in untreated CRC, and joint high expression of the stromal signatures predicted resistance to radiotherapy in rectal cancer. These data show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stromal component.

Ultrasonographically-guided fine-needle aspiration of axillary lymph nodes: role in breast cancer management

The knowledge of the status of axillary lymph nodes (LN) of patients with breast cancer is a fundamental prerequisite in the therapeutic decision. In the present work, we evaluated the impact of fine-needle aspiration cytology (FNAC) of ultrasonographically (US) selected axillary LN in the diagnosis of LN metastases and subsequently in the treatment of patients with breast cancer. Axillary US was performed in 298 patients with diagnosed breast cancer (267 invasive carcinomas and 31 ductal carcinoma in situ DCIS), and in 95 patients it was followed by FNAC of US suspicious LN. Smears were examined by routine cytological staining. Cases of uncertain diagnosis were stained in immunocytochemistry (ICC) with a combination of anticytokeratin and anti-HMFG2 antibodies. Eighty-five FNAC were informative (49 LN were positive for metastases, 36 were negative). In 49 of 267 patients with invasive breast carcinoma (18%), a preoperative diagnosis of metastatic LN in the axilla could be confirmed. These patients could proceed directly to axillary dissection. In addition, US-guided FNAC presurgically scored 49 out of 88 (55%) metastatic LN. Of all others, with nonsuspicious LN on US (203 cases including 31 DCIS), in which no FNAC examination was performed, 28 invasive carcinomas (16%) turned out to be LN positive on histological examination. Based on these data, US examination should be performed in all patients with breast cancer adding ICC-supported FNAC only on US-suspect LN. This presurgical protocol is reliable for screening patients with LN metastases that should proceed directly to axillary dissection or adjuvant chemotherapy, thus avoiding sentinel lymph node biopsy.

Ghrelin in Fetal Thyroid and Follicular Tumors and Cell Lines : Expression and Effects on Tumor Growth

Ghrelin, a growth hormone-releasing hormone produced by gastroenteropancreatic endocrine cells, hypothalamus, and pituitary, was recently identified in medullary thyroid carcinomas and derived cell lines. However, no data exist on its expression in either normal or neoplastic thyroid follicular cells. We analyzed ghrelin expression by immunohistochemistry, in situ hybridization, and reverse transcriptase-polymerase chain reaction in 15 fetal, 4 infant, and 10 adult thyroids, and in 54 tumors of follicular origin. We also analyzed the effects of ghrelin on cell proliferation in N-PAP and ARO thyroid carcinoma cell lines. Ghrelin-binding sites were investigated using reverse transcriptase-polymerase chain reaction to detect its growth hormone secretagogue receptor (GHS-R) mRNA and an in situ-binding localization procedure. Strong ghrelin immunoreactivity was found in fetal but not in infant or adult thyroids. Ghrelin protein and mRNA were present, in variable amounts, in benign and malignant tumors. Normal thyroids, thyroid tumors, and cell lines showed ghrelin binding sites by binding localization, in the absence of the specific GHS receptor mRNA (with the exception of one normal thyroid). Moreover, ghrelin induced dose-dependent inhibition of growth in cell lines. In conclusion, ghrelin is expressed in fetal but not in adult thyroid, and is re-expressed in tumors; the presence of ghrelin receptors other than GHS-R in normal and neoplastic adult thyroid is suggested; ghrelin inhibits cell proliferation of thyroid carcinoma cell lines in vitro.

The Antiproliferative Effect of Synthetic Peptidyl GH Secretagogues in Human CALU-1 Lung Carcinoma Cells

The specific binding of [125I]Tyr-Ala-hexarelin, a radiolabeled peptidyl GH secretagogue (GHS), has been investigated in nontumoral and neoplastic human lung tissues. This binding was very marked in nonendocrine lung carcinomas with values that were greater than found in either normal lung or in endocrine lung neoplasms. Tyr-Ala-hexarelin binding was also present in a human lung carcinoma cell line (CALU-1). [125I]Tyr-Ala-hexarelin binding to tumor membranes was displaced by peptidyl GHS (GHRP-6, hexarelin) and EP-80317, an hexarelin analog devoid of GH-releasing activity in vivo. In contrast, no competition was observed in the presence of the nonpeptidyl GHS MK-0677 and the endogenous ligand of the GHS-R1a ghrelin. GHS-R1a mRNA expression was found in 50% of endocrine lung tumors but was never seen in other nontumoral and neoplastic lung tissues nor in CALU-1. In these cells, hexarelin and EP-80317, but not ghrelin or MK-0677, caused a dose-dependent inhibition of IGF-II-stimulated thymidine incorporatio...

PAF Produced by Human Breast Cancer Cells Promotes Migration and Proliferation of Tumor Cells and Neo-Angiogenesis

Platelet-activating factor (PAF), a phospholipid mediator of inflammation, is present in breast cancer tissue and correlates with microvessel density. In the present study, we investigated the biological significance of PAF synthesized within breast cancer. In vitro, we observed the production of PAF by two estrogen-dependent (MCF7 and T-47D) and an estrogen-independent (MDA-MB231) breast cancer cell lines after stimulation with vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, tumor necrosis factor, thrombin but not with estrogen, progesterone, and oxytocin. The sensitivity to agonist stimulation and the amount of PAF synthesized as cell-associated or released varied in different cell lines, being higher in MDA-MB231 cells, which are known to be highly invasive. We further demonstrate, by reverse transcriptase-polymerase chain reaction and cytofluorimetry, that all of the breast cancer cells express the PAF receptor and respond to PAF stimulation in terms of proliferation. Moreover, in MDA-MB231 cells PAF elicited cell motility. In vivo, two structurally different PAF receptor antagonists WEB 2170 and CV 3988 significantly reduced the formation of new vessels in a tumor induced by subcutaneous implantation of MDA-MB231 cells into SCID mice. In conclusion, these results suggest that PAF, produced and released by breast cancer cells, can contribute to tumor development by enhancing cell motility and proliferation and by stimulating the angiogenic response.

Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its localization in caveolin-1 enriched domains

We have recently shown that oxytocin inhibits cell proliferation when the vast majority of oxytocin receptors are excluded from caveolin-1-enriched microdomains, and that, on the contrary, it has a mitogenic effect when the receptors are targeted to these plasma membrane domains. In this study, we investigated whether the receptors located inside and outside caveolar microdomains initiate different signalling pathways and how this may lead to opposite effects on cell proliferation. Our data indicate that, depending on their localization, oxytocin receptors transactivate EGFR and activate ERK1/2 using different signalling intermediates. The final outcome is a different temporal pattern of EGFR and ERK1/2 phosphorylation, which is more persistent when the receptors are located outside caveolar microdomains and inhibit cell growth, and very transient when they are located in caveolar microdomains and stimulate cell growth. Finally, only the activation of receptors located outside caveolar microdomains correlates with the activation of the cell cycle inhibitor p21WAF1/CIP1, thus suggesting that the antiproliferative OTR effects may, in this case, be achieved by a sustained activation of EGFR and MAPK leading to the induction of this cell cycle regulator.

Expression of apocrine differentiation markers in neuroendocrine breast carcinomas of aged women

Neuroendocrine (NE) breast carcinomas are a rare entity in young women; however, their frequency increases in aged patients. The present work demonstrates that NE breast carcinomas in elderly women can also express an apocrine immunophenotype and analyzes the histological and clinical aspects of such differentiation. A selected series of 50 NE tumors (positive for NE markers in ≥50% of the cells) was tested for the immunocytochemical expression of gross cystic disease fluid protein-15 (GCDFP-15). The results demonstrated that about 50% of moderately (G2) and well-differentiated (G1) NE breast carcinomas (mucinous, solid papillary, and solid cohesive histotypes) coexpressed the apocrine marker. In these cases, specific mRNA for GCDFP-15 (PIP) and for chromogranin A (ChA) was demonstrated using in situ hybridization (ISH). Carcinomas of the alveolar subtype (G2) and poorly differentiated carcinomas (G3), including one case of atypical carcinoid, were pure NE carcinomas, devoid of apocrine differentiation. The steroid receptor status of these lesions was evaluated to test a possible involvement of androgen receptors in apocrine differentiation. We demonstrated that the level of AR and the mean age of patients at diagnosis were significantly higher in apocrine than in nonapocrine differentiated tumors. The histological grade and the expression of estrogen receptor (ER) significantly influenced the prognosis of these NE carcinomas, either pure or NE-apocrine differentiated. The most original result of our study is therefore the demonstration of a possible divergent apocrine differentiation of NE breast carcinomas that might be regulated by the activation of androgen receptors in elder patients. In addition, the possibility for using Chs or GCDFP-15 serum values in the follow-up of these patients, as demonstrated in two cases of the present series, can justify the immunophenotyping of the tumors.

Localization of the human oxytocin receptor in caveolin-1 enriched domains turns the receptor-mediated inhibition of cell growth into a proliferative response.

In this study, we investigated the functional role of the localization of human OTR in caveolin-1 enriched membrane domains. Biochemical fractionation of MDCK cells stably expressing the WT OTR-GFP indicated that only minor quantities of receptor are partitioned in caveolin-1 enriched domains. However, when fused to caveolin-2, the OTR protein proved to be exclusively localized in caveolin-1 enriched fractions, where it bound the agonist with increased affinity and efficiently coupled to Gαq/11. Interestingly, the chimeric protein was unable to undergo agonist-induced internalization and remained confined to the plasma membrane even after prolonged agonist exposure (120 min). A striking difference in receptor stimulation was observed when the OT-induced effect on cell proliferation was analysed: stimulation of the human WT OTR inhibited cell growth, whereas the chimeric protein had a proliferative effect. These data indicate that the localization of human OTR in caveolin-1 enriched microdomains radically alters its regulatory effects on cell growth; the fraction of OTR residing in caveolar structures may therefore play a crucial role in regulating cell proliferation.

OXYTOCIN INHIBITS THE PROLIFERATION OF MDA-MB231 HUMAN BREAST-CANCER CELLS VIA CYCLIC ADENOSINE MONOPHOSPHATE AND PROTEIN KINASE A

Oxytocin (OT) inhibits the proliferation of breast‐cancer cells in vitro via a specific G‐coupled receptor. To elucidate the intracellular mechanism involved in this biological effect, different G‐coupled receptor mediators have been investigated in untreated and OT‐treated MDA‐MB231 breast‐carcinoma cells. In these cells, after OT treatment, a significant cAMP increase was observed using a radioimmunoassay procedure, whereas the Ca2+ (determined with the fluorescent probe fura‐2) and the inositol phosphate (determined after cell labeling with myo(2‐3H)‐inositol) concentrations were not modified, contrary to what has been observed in myometrial and myo‐epithelial cells. The PKA inhibitor PKI (6‐22) amide reverted the effect of OT, indicating that the anti‐proliferative effect of the peptide is strictly related to the cAMP–PKA pathway. OT treatment did not modify tyrosine phosphorylation either. Our results indicate that in breast epithelial cells devoid of contractile activity, cAMP is the intracellular mediator of OT action, whereas the Ca2+‐phosphoinositide system is not involved. Int. J. Cancer 72:340–344, 1997.

Routine assessment of prognostic factors in breast cancer using a multicore tissue microarray procedure

We propose multicore tissue microarray (TMA) as an alternative to whole section for routine assessment of prognostic factors in breast cancer. Since 2004, we introduced the multicore TMA for testing estrogen (ER) and progesterone receptors (PR), proliferation activity by Ki67, and HER2 overexpression and amplification in routine work. At least four tumor foci were selected on the whole section, and a dedicated technician used a stereomicroscope for accurate sampling of the selected areas. To identify a specific case in the TMA, a separate file and a computerized reporting form with the TMA map were created. A preliminary pilot study comparing the TMA results with those obtained on whole sections showed the specificity of the procedure. Moreover, in everyday diagnosis, hormone receptors were repeated on full section when negative in TMA, without significant discrepancy. Retrospective analysis of the 237 breast carcinomas studied by TMA showed the expected correspondence of tumor-grade differentiation with the hormone receptor pattern, the proliferation activity, and HER2 immunohistochemical and FISH values. In conclusion, multicore TMA may be an efficient approach in the routine study of prognostic factors in breast cancer, significantly reducing costs, time, and burden of slides necessary to accomplish these mandatory tests.