Daniel F. Alonso

Reduction of mouse mammary tumor formation and metastasis by lovastatin, an inhibitor of the mevalonate pathway of cholesterol synthesis

Lovastatin, a fungal antibiotic used in the treatment of hypercholesterolemia, is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key regulatory enzyme in the mevalonate pathway of cholesterol synthesis. We examined the antitumor properties of lovastatin on the F3II sarcomatoid mammary carcinoma, a highly invasive and metastatic murine tumor model. Female BALB/c inbred mice were inoculated subcutaneously with F3II tumor cells and injected i.p. daily with 10 mg/kg body weight of lovastatin or administered p.o. at a level corresponding to the human dosage of 1–2 mg/kg/day. Treatment significantly prolonged tumor latency and reduced tumor formation and metastatic dissemination to the lungs from established mammary tumors. In vitro, antitumor properties of lovastatin were strongly associated with inhibition of tumor cell attachment and migration. These actions were prevented by addition of mevalonate but not by equivalent concentrations of farnesyl pyrophosphate. In accordance, Western blot assays showed that lovastatin effects did not appear to be related to modifications in Ras oncoproteins in our model. The present data indicate that lovastatin could be an antitumor agent with potentially useful clinical applications in breast cancer.

CIGB-300, a novel proapoptotic peptide that impairs the CK2 phosphorylation and exhibits anticancer properties both in vitro and in vivo

Protein Kinase CK2 is a serine-threonine kinase frequently deregulated in many human tumors. Here, we hypothesized that a peptide binder to the CK2 phosphoacceptor site could exhibit anticancer properties inxa0vitro, in tumor animal models, and in cancer patients. By screening a random cyclic peptide phage display library, we identified the CIGB-300 (formerly P15-Tat), a cyclic peptide which abrogates the CK2 phosphorylation by blocking recombinant substrates inxa0vitro. Interestingly, synthetic CIGB-300 led to a dose-dependent antiproliferative effect in a variety of tumor cell lines and induced apoptosis as evidenced by rapid caspase activation. Importantly, CIGB-300 elicited significant antitumor effect both by local and systemic administration in murine syngenic tumors and human tumors xenografted in nude mice. Finally, we performed a First-in-Man trial with CIGB 300 in patients with cervical malignancies. The peptide was found to be safe and well tolerated in the dose range studied. Likewise, signs of clinical benefit were clearly identified after the CIGB-300 treatment as evidenced by significant decrease of the tumor lesion area and histological examination. Our results provide an early proof-of-principle of clinical benefit by using an anti-CK2 approach in cancer. Furthermore, this is the first clinical trial where an investigational drug has been used to target the CK2 phosphorylation domain.

Safety and preliminary efficacy data of a novel Casein Kinase 2 (CK2) peptide inhibitor administered intralesionally at four dose levels in patients with cervical malignancies

BackgroundCervical cancer is now considered the second leading cause of death among women worldwide, and its incidence has reached alarming levels, especially in developing countries. Similarly, high grade squamous intraepithelial lesion (HSIL), the precursor stage for cervical cancer, represents a growing health problem among younger women as the HSIL management regimes that have been developed are not fully effective. From the etiological point of view, the presence of Human Papillomavirus (HPV) has been demonstrated to play a crucial role for developing cervical malignancies, and viral DNA has been detected in 99.7% of cervical tumors at the later stages. CIGB-300 is a novel cyclic synthetic peptide that induces apoptosis in malignant cells and elicits antitumor activity in cancer animal models. CIGB-300 impairs the Casein Kinase (CK2) phosphorylation, by targeting the substrates phosphoaceptor domain. Based on the perspectives of CIGB-300 to treat cancer, this first-in-human study investigated its safety and tolerability in patients with cervical malignancies.MethodsThirty-one women with colposcopically and histologically diagnosed microinvasive or pre-invasive cervical cancer were enrolled in a dose escalating study. CIGB-300 was administered sequentially at 14, 70, 245 and 490 mg by intralesional injections during 5 consecutive days to groups of 7 – 10 patients. Toxicity was monitored daily until fifteen days after the end of treatment, when patients underwent conization. Digital colposcopy, histology, and HPV status were also evaluated.ResultsNo maximum-tolerated dose or dose-limiting toxicity was achieved. The most frequent local events were pain, bleeding, hematoma and erythema at the injection site. The systemic adverse events were rash, facial edema, itching, hot flashes, and localized cramps. 75% of the patients experienced a significant lesion reduction at colposcopy and 19% exhibited full histological regression. HPV DNA was negative in 48% of the previously positive patients. Long term follow-up did not reveal recurrences or adverse events.ConclusionCIGB 300 was safe and well tolerated. This is the first clinical trial where a drug has been used to target the CK2 phosphoaceptor domain providing an early proof-of-principle of a possible clinical benefit.

CIGB-300, a synthetic peptide-based drug that targets the CK2 phosphoaceptor domain. Translational and clinical research

CK2 represents an oncology target scientifically validated. However, clinical research with inhibitors of the CK2-mediated phosphorylation event is still insufficient to recognize it as a clinically validated target. CIGB-300, an investigational peptide-based drug that targets the phosphoaceptor site, binds to a CK2 substrate array in vitro but mainly to B23/nucleophosmin in vivo. The CIGB-300 proapoptotic effect is preceded by its nucleolar localization, inhibition of the CK2-mediated phosphorylation on B23/nucleophosmin and nucleolar disassembly. Importantly, CIGB-300 shifted a protein array linked to apoptosis, ribosome biogenesis, cell proliferation, glycolisis, and cell motility in proteomic studies which helped to understand its mechanism of action. In the clinical ground, CIGB-300 has proved to be safe and well tolerated in a First-in-Human trial in women with cervical malignancies who also experienced signs of clinical benefit. In a second Phase 1 clinical trial in women with cervical cancer stage IB2/II, the MTD and DLT have been also identified in the clinical setting. Interestingly, in cervical tumors the B23/nucleophosmin protein levels were significantly reduced after CIGB-300 treatment at the nucleus compartment. In addition, expanded use of CIGB-300 in case studies has evidenced antitumor activity when administered as compassional option. Collectively, our data outline important clues on translational and clinical research from this novel peptide-based drug reinforcing its perspectives to treat cancer and paving the way to validate CK2 as a promising target in oncology.

Antimetastatic effect of desmopressin in a mouse mammary tumor model

We have investigated the effects of desmopressin (DDAVP), a synthetic analog of the natural hormone vasopressin, on experimental lung colonization of mammary tumor cells using a syngeneic BALB/c mouse model. Coinjection of DDAVP (1–2u2009μg/kg body weight) at the time of i.v. inoculation of F3II carcinoma cells or LM3 adenocarcinoma cells significantly inhibited the formation of experimental lung metastases. In both cases, the number of pulmonary nodules was reduced about 70%. Inhibition of metastasis was also obtained with i.v. administration of DDAVP 24u2009h after tumor cell inoculation. Interestingly, the inhibition of lung metastasis was not due to direct cytotoxic effects of DDAVP on mammary tumor cells. The in vitro formation of multicellular aggregates in the presence of citrated plasma from control and DDAVP‐treated mice was also examined. Control plasma rapidly induced a significant tumor cell aggregation. In contrast, in the presence of plasma from DDAVP‐treated mice, tumor cells remained as a single cell suspension. DDAVP may help to dissolve the protective fibrin shield of circulating tumor cells. Our data suggest, for the first time, that adjuvant DDAVP therapy may impair successful implantation of circulating mammary tumor cells.

Chronic in vitro exposure to 3'-azido-2', 3'-dideoxythymidine induces senescence and apoptosis and reduces tumorigenicity of metastatic mouse mammary tumor cells.

Normal cells in culture divide a certain amount of times and undergo a process termed replicative senescence. Telomere loss is thought to control entry into senescence. Activation of telomerase in tumors bypasses cellular senescence and is thus a requirement for tumor progression. We reported previously the preferential incorporation of 3′-azido-2′, 3′-dideoxythymidine (AZT) in telomeric sequences of immortalized cells in culture. In this work, we have investigated the effects of chronic in vitro AZT exposure on F3II mouse mammary carcinoma cells. We demonstrate, for the first time, that AZT-treated tumor cells have a reduced tumorigenicity in syngeneic BALB/c mice. Tumor incidence was reduced and survival was prolonged in animals inoculated with AZT-treated cells when comparing with control counterparts. The number and size of spontaneous metastases were also decreased in animals inoculated with AZT-treated cells. In addition, we present evidence of morphological and biochemical signs of senescence, as shown by the staining for senescence associated ß-galactosidase activity, and induction of programmed cell death, as demonstrated by an increase of caspase-3 activity, in tumor cells exposed to AZT. These data indicate that chronic exposure of mammary carcinoma cells to AZT may be sufficient to induce a senescent phenotype and to reduce tumorigenicity.

MODULATION OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR AND METALLOPROTEINASE ACTIVITIES IN CULTURED MOUSE MAMMARY-CARCINOMA CELLS: ENHANCEMENT BY PACLITAXEL AND INHIBITION BY NOCODAZOLE

Paclitaxel is a potent anti‐tumor drug used in the treatment of breast cancer. It induces de‐centralization of the microtubular system in tumor cells, blocking cell division. In the search for dissemination to a secondary site, cancer cells are capable of degrading most components of the extracellular matrix via an extracellular proteolytic cascade, including urokinase‐type plasminogen activator (uPA) and the matrix metalloproteinases (MMPs). In the present study, the effects of paclitaxel and nocodazole, 2 drugs known to affect microtubules with opposite mechanisms of action, have been tested for their effect on the secretion of uPA and MMPs in cultures of F3II mouse mammary‐tumor cells. Tumor‐derived uPA activity significantly increased after pre‐treatment of tumor cells for 24 hr with micromolar concentrations of paclitaxel (4 μM), while decreasing after pre‐treatment with nocodazole (1 μM). A similar modulation was found for MMP‐9 by zymographic analysis. Immunofluorescence and Western‐blot analysis confirmed the formation of parallel microtubule fragments in paclitaxel‐treated cells and almost complete de‐polymerization of microtubules in nocodazole‐treated ones. Our data suggest that, through opposite actions on microtubule organization and dynamics, paclitaxel and nocodazole exert inverse modulation of tumor‐derived proteolytic activity in mammary tumor cells. Int. J. Cancer 83:242–246, 1999.

A phase I study of the anti-idiotype vaccine racotumomab in neuroblastoma and other pediatric refractory malignancies

Pediatric neuroectodermal malignancies express N‐glycolylated gangliosides including N‐glycolyl GM3 (NeuGcGM3) as targets for immunotherapy.

Pharmacological inhibition of Rac1-PAK1 axis restores tamoxifen sensitivity in human resistant breast cancer cells

Tamoxifen is a standard endocrine therapy for estrogen receptor positive breast cancer patients. Despite its success, development of resistance mechanisms is still a serious clinical problem. Deregulation of survival signaling pathways play a key role in tamoxifen resistance, being upregulation of Rac1-PAK1 signaling pathway one of the most important. Here, we report the development of the breast cancer cell model MCF7::C1199 having Rac1 enhanced activity with the aim of evaluating the role of Rac1 in acquired endocrine resistance. These cells not only showed distinctive features of Rac1-regulated process as increased migration and proliferation rates, but also showed that upregulation of Rac1 activity triggered a hormonal-independent and tamoxifen resistant phenotype. We also demonstrated that PAK1 activity increases in response to Tamoxifen, increasing phosphorylation levels of estrogen receptor at Ser305, a key phosphorylation site involved in tamoxifen resistance. Finally, we evaluated the effect of 1A-116, a specific Rac1 inhibitor developed by our group, in tamoxifen-resistant cells. 1A-116 effectively restored tamoxifen anti-proliferative effects, switched off PAK1 activity and decreased estrogen receptor phospho-Ser305 levels. Since combination schemes of novel targeted agents with endocrine therapy could be potential new strategies to restore tamoxifen sensibility, these results show that inhibition of Rac1-PAK1 signaling pathway may provides benefits to revert resistance mechanisms in endocrine therapies.

Role of Tumor-Derived Granulocyte-Macrophage Colony-Stimulating Factor in MiceBear ing a Highly Invasive and Metastatic Mammary Carcinoma

We have examined the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in tumor-bearing BALB/c mice using the syngeneic F3II mammary carcinoma. In the present model, progression of subcutaneous tumors induced massive myelopoiesis in bone marrow and spleen due to GM-CSF secretion by tumor cells. In vitro, the addition of recombinant mouse GM-CSF (5– 25 ng/ml) caused a significant increase in F3II cell growth, either in the presence or absence of serum. Zymographic analysis of conditioned media from F3II monolayers showed that GM-CSF exerted a dose-dependent enhancement in the metalloproteinases MMP-9 (105 kD) and MMP-2 (70 kD), key enzymes in mammary tumor cell invasion. Our data suggest that ectopic GM-CSF production stimulates myelopoiesis and may also play an important role in tumor progression and metastasis formation.