Emanuela Pilozzi

Identification and expansion of human colon-cancer-initiating cells

Colon carcinoma is the second most common cause of death from cancer. The isolation and characterization of tumorigenic colon cancer cells may help to devise novel diagnostic and therapeutic procedures. Although there is increasing evidence that a rare population of undifferentiated cells is responsible for tumour formation and maintenance, this has not been explored for colorectal cancer. Here, we show that tumorigenic cells in colon cancer are included in the high-density CD133+ population, which accounts for about 2.5% of the tumour cells. Subcutaneous injection of colon cancer CD133+ cells readily reproduced the original tumour in immunodeficient mice, whereas CD133- cells did not form tumours. Such tumours were serially transplanted for several generations, in each of which we observed progressively faster tumour growth without significant phenotypic alterations. Unlike CD133- cells, CD133+ colon cancer cells grew exponentially for more than one year in vitro as undifferentiated tumour spheres in serum-free medium, maintaining the ability to engraft and reproduce the same morphological and antigenic pattern of the original tumour. We conclude that colorectal cancer is created and propagated by a small number of undifferentiated tumorigenic CD133+ cells, which should therefore be the target of future therapies.

Co-expression of CD79a (JCB117) and CD3 by lymphoblastic lymphoma

Acute lymphoblastic leukaemia/lymphoma is a malignant disorder derived from the clonal proliferation of lymphoid precursor cells. Whether the tumour cells are of B‐ or T‐cell type is an important criterion for prognosis which has not been available previously to pathologists, due to the lack of a reliable early B‐cell marker functioning on routinely processed material. This has changed with the production of monoclonal antibodies against the B‐cell signalling molecule CD79a. CD79a is expressed on normal and neoplastic B cells from the early stages of B‐cell maturation and has been considered to be B‐cell‐specific. Currently available antibodies against CD79a, in particular JCB117, allow the identification of B cells, and hence B lymphoblastic disease, in paraffin‐embedded material. In this study, the expression of CD79a (JCB117) and CD3 has been investigated in 149 cases of T and 68 cases of B lymphoblastic leukaemia/ lymphoma. For the first time, co‐expression of CD79a (JCB117) and CD3 is reported in 10 per cent of cases of T lymphoblastic leukaemia/lymphoma. This finding raises questions about the co‐expression of T‐ and B‐cell markers in the development of lymphocytes, benign as well as malignant, and alerts pathologists to a potential problem in diagnosis. Copyright

Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.

Type I gastric carcinoids: a prospective study on endoscopic management and recurrence rate.

Background: Type I gastric carcinoids (TIGCs) are neuroendocrine neoplasms arising from enterochromaffin-like cells in atrophic body gastritis. Data regarding their evolution in prospective series are scarce, thus treatment and follow-up are not codified. Our aim was to evaluate clinical outcome and recurrence in TIGCs managed by endoscopic approach. Methods: 33 patients (24 females; median age 65 years, range 23–81) were included and managed through endoscopic follow-up every 6–12 months, with lesion removal and multiple gastric biopsies. Baseline clinical and histological features were analyzed as risk factors by Cox regression. Results: At diagnosis, 7 tumors were intramucosal carcinoids and 26 were polyps (median diameter 5 mm, range 2–20), multiple in 17 patients. Associated severe atrophy was present in 21 cases (63.6%), while mild atrophy was found in 6 cases (18.2%). During a 46-month median follow-up, survival was 100% and no metastases occurred. One patient developed a less-differentiated carcinoid that was radically treated by surgery. 21 patients (63.6%) had recurrence after a median of 8 months, 14 of these (66.6%) had a second recurrence after a median of 8 months following the previous carcinoid removal. Median recurrence-free survival was 24 months. Neither clinical nor biochemical recurrence-predicting factors were found. Conclusions: Although about 60% of TIGCs had recurrence after endoscopic resection, endoscopic management may be considered safe and effective.

Development of type I gastric carcinoid in patients with chronic atrophic gastritis

Background  Long‐term observational studies assessing the incidence of type I gastric carcinoid (typeIGC) in patients with chronic atrophic gastritis are few.

Gene rearrangements in T-cell lymphoblastic lymphoma.

This study presents an examination of the Ig heavy chain (IgH) and T‐cell receptor gamma (TCRγ) genes in a series of 39 CD3‐positive T‐cell acute lymphoblastic leukaemia (ALL) cases with and without co‐expression of CD79a; 30/39 cases had a rearrangement of the TCRγ genes and two of these 30 cases also demonstrated an IgH rearrangement. No cases had solely an IgH rearrangement. The conclusion of the study is that lymphoblastic lymphoma cases that are positive for CD3 are of T‐cell lineage, regardless of CD79a expression. Copyright

Alteration of local microflora and α-defensins hyper-production in colonic adenoma mucosa

Background and Aim Gut flora/host interactions are fundamental for the maintenance of homeostasis. Evidence of possible regulatory effect of commensal bacteria on proliferative disorders of the colon is mounting. In this study, we explored the hypothesis that precancerous lesions, such as adenomas, present alteration of the local microflora and lead to an overproduction of antibacterial molecules of the innate immunity, namely &agr;-defensins. Thus, the host-bacteria misbalance could represent a potential procarcinogenic factor. Methods Biopsies from adenomatous polyps and normal mucosa, in the rectum-sigmoid colon, were collected from 51 patients. Concentration of mucosal bacteria was evaluated by real-time polymerase chain reaction after extraction of total DNA. Total RNA was also extracted, and the defensin &agr;-1, defensin-5, and defensin-6 gene expressions were evaluated by real-time polymerase chain reaction. Immunohistochemical study has been carried out to evaluate protein production and location. Antibacterial activity of adenomatous polyps mucosa was evaluated in vitro. Results Biopsies from adenomatous polyps had a significant relative reduction of mucosa adherent bacteria compared with normal tissue (20-fold relative reduction, P<0.05). Concomitantly, &agr;-defensin expression and production were significantly increased in adenomas. Adenoma mucosa showed increased antibacterial activity in vitro compared with normal mucosa. Conclusions Microflora dysbiosis occurs at the mucosal surface in colonic adenomas, and may represent a potential factor for dysplastic cell proliferation. Further studies are needed to confirm and define the role of this mechanism in colon carcinogenesis and the potential applications in the clinical setting.

Thymosin β4 targeting impairs tumorigenic activity of colon cancer stem cells

Thymosin β4(Tβ4) is an actin‐binding peptide overexpressed in several tumors, including colon carcinomas. The aim of this study was to investigate the role of Tβ4 in promoting the tumorigenic properties of colorectal cancer stem cells (CR‐CSCs), which are responsible for tumor initiation and growth. We first found that CR‐CSCs from different patients have higher Tβ4 levels than normal epithelial cells. Then, we used a lentiviral strategy to down‐regulate Tβ4 expression in CR‐CSCs and analyzed the effects of such modulation on proliferation, survival, and tumorigenic activity of CR‐CSCs. Empty vector‐transduced CR‐CSCs were used as a control. Targeting of the Tβ4 produced CR‐CSCs with a lower capacity to grow and migrate in culture and, interestingly, reduced tumor size and aggressiveness of CR‐CSC‐based xenografts in mice. Moreover, such loss in tumorigenic activity was accompanied by a significant increase of phosphatase and tensin homologue (PTEN) and a concomitant reduction of the integrin‐linked kinase (ILK) expression, which resulted in a decreased activation of protein kinase B (Akt). Accordingly, exogenous expression of an active form of Akt rescued all the protumoral features lost after Tβ4 targeting in CR‐CSCs. In conclusion, Tβ4 may have important implications for therapeutic intervention for treatment of human colon carcinoma.—Ricci‐Vitiani, L., Mollinari, C., di Martino, S., Biffoni, M., Pilozzi, E., Pagliuca, A., Chiara de Stefano, M., Circo, R., Merlo, D., De Maria, R., Garaci, E. Thymosin β4 targeting impairs tumorigenic activity of colon cancer stem cells. FASEB J. 24, 4291–4301 (2010). www.fasebj.org

Src kinase activity coordinates cell adhesion and spreading with activation of mammalian target of rapamycin in pancreatic endocrine tumour cells.

Pancreatic endocrine tumours (PETs) are rare and heterogeneous neoplasms, often diagnosed at metastatic stage, for which no cure is currently available. Recently, activation of two pathways that support proliferation and invasiveness of cancer cells, the Src family kinase (SFK) and mammalian target of rapamycin (mTOR) pathways, was demonstrated in PETs. Since both pathways represent suitable targets for therapeutic intervention, we investigated their possible interaction in PETs. Western blot and immunofluorescence analyses indicated that SFK and mTOR activity correlate in PET cell lines. We also found that SFKs coordinate cell adhesion and spreading with activation of the mTOR pathway in PET cells. Live cell metabolic labelling and biochemical studies demonstrated that SFK activity enhance mTOR-dependent translation initiation. Furthermore, microarray analysis of the mRNAs associated with polyribosomes revealed that SFKs regulate mTOR-dependent translation of specific transcripts, with an enrichment in mRNAs encoding cell cycle proteins. Importantly, a synergic inhibition of proliferation was observed in PET cells concomitantly treated with SFK and mTOR inhibitors, without activation of the phosphatidylinositol 3-kinase/AKT pro-survival pathway. Tissue microarray analysis revealed activation of Src and mTOR in some PET samples, and identified phosphorylation of 4E-BP1 as an independent marker of poor prognosis in PETs. Thus, our work highlights a novel link between the SFK and mTOR pathways, which regulate the translation of mRNAs for cell cycle regulators, and suggest that crosstalk between these pathways promotes PET cell proliferation.

Advanced digestive neuroendocrine tumors: Metastatic pattern is an independent factor affecting clinical outcome

Objectives The objective of this study was to determine the impact of different metastatic spread patterns on outcome in advanced digestive neuroendocrine tumors (NETs). Methods This was a retrospective analysis of patients with stage IV NETs, classified as group 1 (unilobar liver metastases), group 2 (bilobar liver metastases), group 3 (extra-abdominal metastases). End points were overall survival (OS) and progression-free survival (PFS). Risk factor analysis was performed using Cox proportional hazard model. Results Of the 229 patients, 135 (58.9%) had pancreatic, and 94 (41.1%) small bowel NETs: 32 (13.9%) were included in group 1, 179 (78.2%) in group 2, and 18 (7.9%) in group 3. Median Ki67 was 4.5%. Overall, 5-year OS was 55%. Different OS was observed among the 3 groups: median survival not reached, 81 and 8 months, respectively (P < 0.001). Median PFS was 18 months. Both OS and PFS were significantly affected by Ki67 and metastatic spread pattern. Conclusions The stratification of stage IV NET patients based on metastatic patterns, alongside Ki67, predicts the clinical outcome. The extent of metastatic disease is a previously unrecognized variable, which should be considered when evaluating the results of treatments in NET patients with advanced disease.