Ramón Vilella

Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs.

Lipid transfer protein syndrome: clinical pattern, cofactor effect and profile of molecular sensitization to plant‐foods and pollens

Multiple plant‐food sensitizations with a complex pattern of clinical manifestations are a common feature of lipid transfer protein (LTP)‐allergic patients. Component‐resolved diagnosis permits the diagnosis of the allergen sensitization profile.

Effect of proteasome inhibitors on proliferation and apoptosis of human cutaneous melanoma-derived cell lines

Background  Cutaneous malignant melanoma is an aggressive type of skin cancer which causes disproportionate mortality in young and middle‐aged adults. Once disseminated, melanoma can be considered an incurable disease, highly resistant to standard antineoplastic treatment, such as chemotherapy or radiation therapy. The proteasome represents a novel target for cancer therapy that can potentially be used in melanoma.

Identification of the amino acid residues defining an intralocus determinant in the α1 domain of HLA-A molecules

A major effort has been made to define the molecular basis of the polymorphism in HLA molecules, but little is known regarding the relevance of the shared antigenic determinants. Nevertheless, the description of conserved regions on molecules controlled by to the same (intralocus) or different (interlocus) HLA loci is important in view of their increasing functional (Taylor et al. 1986), structural, and phylogenetic (Parham et al. 1988) significance. Mouse monoclonal antibodies (mAbs) specific for human HLA class I antigens often react with public determinants. Serological (Spear et al. 1985, Yang et al. 1984) and molecular (Parham et al. 1988) evidence for the existence of interand intralocus determinants in the human HLA system has been reported. Progress in gene cloning, exon shuffling, and crystallography of HLA class I antigens is now permitting the molecular mapping of some of these epitopes on to specific residues of the HLA external domains. Some information about HLA epitopes can also be gained by testing the reactivity of mAbs against either natural or in vitro-generated (Santos-Aguado et al. 1988) HLA mutant molecules. In the present study we characterize serologically and biochemically a new mAb (1082C5) produced in our laboratory which recognizes an intralocus determinant present on a limited number of HLA-A locus-encoded gene products (HLA-A2, -A3, -A28, -A29, -A30, -A31 and -A33). Furthermore, by testing its reactivity with HLA-A2 natural variants and oligonucleotide-generated, site-directed HLA-A2 mutants, we also provide evidence for the importance of amino acid residues 79 and/or 80 of the ~1 domain in the formation of such an intralocus HLA-A determinant. The HLA class I specificity of the 1082C5 mAb was clearly demonstrated by its reactivity with cells and by

Inhibition of activated receptor tyrosine kinases by Sunitinib induces growth arrest and sensitizes melanoma cells to Bortezomib by blocking Akt pathway

Despite the use of multiple therapeutic strategies, metastatic melanoma remains a challenge for oncologists. Thus, new approaches using combinational treatment may be used to try to improve the prognosis of this disease. In this report, we have analyzed the expression of receptor tyrosine kinases (RTKs) in melanoma specimens and in four metastatic melanoma cell lines. Both melanoma specimens and cell lines expressed RTKs, suggesting that they may represent eventual targets for multitargeted tyrosine kinase inhibitor, Suntinib. Sunitinib reduced the proliferation of two melanoma cell lines (M16 and M17) and increased apoptosis in one of them (M16). Moreover, the two metastatic melanoma cell lines harbored an activated receptor (PDGFRα and VEGFR, respectively), and Sunitinib suppressed the phosphorylation of the RTKs and their downstream targets Akt and ribosomal protein S6, in these two cell lines. Similar results were obtained when either PDGFRα or VEGFR2 expression was silenced by lentiviral‐mediated short‐hairpin RNA delivery in M16 and M17, respectively. To evaluate the interaction between Sunitinib and Bortezomib, median dose effect analysis using MTT assay was performed, and combination index was calculated. Bortezomib synergistically enhanced the Sunitinib‐induced growth arrest in Sunitinib‐sensitive cells (combination index < 1). Moreover, LY294002, a PI3K inhibitor, sensitized melanoma cells to Bortezomib treatment, suggesting that downregulation of phospho‐Akt by Sunitinib mediates the synergy obtained by Bortezomib + Sunitinib cotreatment. Altogether, our results suggest that melanoma cells harboring an activated RTK may be clinically responsive to pharmacologic RTK inhibition by Sunitinib, and a strategy combining Sunitinib and Bortezomib, may provide therapeutic benefit.

T-type calcium channel blockers inhibit autophagy and promote apoptosis of malignant melanoma cells.

We have recently reported that human melanoma cells express a variety of voltage‐gated calcium (Ca2+) channel types, including low‐voltage‐activated T‐type channels that play a significant role in melanoma cell cycle progression. Here, we challenged melanoma metastatic cells with T‐type channel blockers of clinical use and found a dual effect on cell viability: (i) a reduction in the proliferation rate, through a halt in the progression to the G1‐S phase; and (ii) a promotion of cell death that was partially dependent on the activation of caspases. An in‐depth analysis of the death process showed that the apoptotic pathway is preceded by endoplasmic reticulum stress and the subsequent inhibition of the basal macroautophagy which is active in these cells. The effects of pharmacological blockers on Ca2+ homeostasis, autophagy, and cell death were mimicked by T‐type channel gene silencing. These results provide the basis for a new pharmacological and/or gene silencing approach toward tackling melanoma metastasis.

Toxicity of combined treatment of adjuvant irradiation and interferon α2b in high-risk melanoma patients

Surgically resected stage III melanoma patients commonly receive adjuvant therapy with interferon (IFN) &agr;2b. For those patients with high-risk features of draining node recurrence, radiation therapy can also be considered as a treatment option. The purpose of this retrospective study was to assess the efficacy and radiation-related toxicity of this combined therapy. Eighteen patients receiving adjuvant IFN&agr;2b therapy during radiation therapy, or within 1 month of its completion, were reviewed retrospectively and analysed for outcome. Radiation was delivered at 600 cGy dose per fraction, in 16 out of 18 patients, twice a week, and at 200 cGy dose per fraction in two patients five times a week. Total radiation dose and number of fractions were as follows: 30 Gy/5 fr (n=8), 36 Gy/6 fr (n=8) and 50 Gy/25 fr (n=2). The percentage of disease-free patients, with no local recurrence, at 3 years was 88%. In 10 patients, IFN&agr;2b was administered concurrently with radiotherapy; in three, within 30 days before or after radiation; and in five, more than 30 days after radiation. All the patients experienced acute skin reactions, grade I on the Radiation Therapy Oncology Group (RTOG) scale. Late radiation-related toxicity was seen in one patient with grade III (RTOG) skin reaction and two with grade IV (RTOG) radiation-induced myelitis. Concurrent use of adjuvant radiotherapy and IFN&agr;2b might enhance radiation-induced toxicity, and special care should be taken when the spinal cord is included in the radiation field.

Treatment of patients with progressive unresectable metastatic melanoma with a heterologous polyvalent melanoma whole cell vaccine

Unresectable metastatic melanoma has no elective treatment. Neither chemotherapy, intravenous IL‐2 nor biochemotherapy clearly improves the overall survival. Recent assays with therapeutic vaccines have been recently yielded promising results. Here, we describe the application, clinical tolerance and antitumoural activity of a heterologous polyvalent melanoma whole cell vaccine in patients with metastatic melanoma. Twenty‐eight AJCC stage III/IV melanoma patients with progressive unresectable metastatic disease were treated with our heterologous polyvalent melanoma whole cell vaccine between July 1, 1998 and July 1, 2002. All patients had already been unsuccessfully treated with high doses of IFN‐α2 and/or polychemotherapy and/or biochemotherapy and/or perfusion of extremities, or could not receive other treatments due to their age or underlying illness. Twenty‐three were assessable. The vaccine was constituted by 10 melanoma cell lines, derived from primary, lymph node and metastatic melanomas. Prior to intradermal inoculation, the cells were irradiated and mixed with BCG, and 50% were treated with DNFB. After a median follow‐up of 19 months, 26% of patients responded: 3 CR (18, 16+, and 26+ months), 2 PR (8 and 22 months) and 1 MR (36+ months). The median survival of the whole group was 20.2 months. None of the 28 patients initially included in the study presented significant toxicity. This vaccination program had specific antitumoural activity in advanced metastatic melanoma patients and was well tolerated. The clinical responses and the median survival of our group of patients, together with the low toxicity of our polyvalent vaccine, suggest that this approach could be applied to earlier metastatic melanoma patients.

Transferrin receptor (CD71) expression in peritoneal macrophages from fertile and infertile women with and without endometriosis.

PROBLEM: Hyperactivated macrophages are implicated in the pathophysiology of endometriosis‐associated infertility. This study investigates transferrin receptor expression (CD71) as a marker of hyperactivity in peritoneal macrophages of infertile patients with minimal to mild endometriosis (group 1, n = 25).

Monoclonal antibody against HLA-Aw32 + A25 is HLA-Aw32 an allele with no unique antigenic determinant?

In the present paper we describe the production and characterization of a monoclonal antibody (Mab) recognizing HLA-Aw32 + A25 antigens. NS1 murine myeloma cells were fused with splenocytes from a BALB/c mouse immunized with normal human peripheral blood (PB) lymphocytes of phenotype A1, Aw32; B7,B37,Cw-,Cw-;DR2,DRw10. Supernatants were first screened against Cr51-labeled immunizing cells by complement dependent cytotoxicity 51Cr-CDC). Cultures identified as producing cytotoxic antibodies were subcultured and the supernatants tested against a selected panel of HLA typed cells by the NIH microcytotoxicity method. One culture producing antibody reacting with an HLA polymorphism was detected. This hybrid, designated CATA 1, was cloned twice by limiting dilution and obtained in ascitic form. Specificity of CATA 1 Mab was evaluated against a panel of 120 PB T cells from normal donors. CATA 1 reacted with cells bearing HLA-A25 or HLA-Aw32 antigens. In addition, a reaction was observed with a cell of phenotype A2,Aw31; B17,Bw49. Isoelectric focusing revealed the monoclonal nature of CATA 1, with immunofixation identifying it as an IgG molecule. Absorption studies have demonstrated that CATA 1 recognizes a common determinant on HLA-A25 and HLA-Aw32. The finding that this Mab recognizes the same CREG as alloantisera against HLA-Aw32 suggests that this antigen has no unique epitopes.