Gene expression analysis of bone metastasis and circulating tumor cells from metastatic castrate-resistant prostate cancer patients.

BACKGROUND: Characterization of genes linked to bone metastasis is critical for identification of novel prognostic or predictive biomarkers and potential therapeutic targets in metastatic castrate-resistant prostate cancer (mCRPC). Although bone marrow core biopsies (BMBx) can be obtained for gene profiling, the procedure itself is invasive and uncommon practice in mCRPC patients. Conversely, circulating tumor cells (CTCs), which are likely to stem from bone metastases, can be isolated from blood. The goals of this exploratory study were to establish a sensitive methodology to analyze gene expression in BMBx and CTCs, and to determine whether the presence or absence of detectable gene expression is concordant in matching samples from mCRPC patients. METHODS: The CellSearch(®) platform was used to enrich and enumerate CTCs. Low numbers of PC3 prostate cancer (PCa) cells were spiked into normal blood to assess cell recovery rate. RNA extracted from recovered PC3 cells was amplified using an Eberwine-based procedure to obtain antisense mRNA (aRNA), and assess the linearity of the RNA amplification method. In this pilot study, RNAs extracted from CTCs and PCa cells microdissected from formalin-fixed paraffin-embedded BMBx, were amplified to obtain aRNA and assess the expression of eight genes functionally relevant to PCa bone metastasis using RT-PCR. RESULTS: RNAs were successfully extracted from as few as 1-5 PCa cells in blood samples. The relative expression levels of reference genes were maintained after RNA amplification. The integrity of the amplified RNA was also demonstrated by RT-PCR analysis using primer sets that target the 5'-end, middle, and 3'-end of reference mRNA. We found that in 21 out of 28 comparisons, the presence or absence of detectable gene expression in CTCs and PCa cells microdissected from single bone lesions of the same patients was concordant. CONCLUSIONS: This exploratory analysis suggests that aRNA amplification through in vitro transcription may be useful as a method to detect gene expression in small numbers of CTCs and tumor cells microdissected from bone metastatic lesions. In some cases, gene expression in CTCs and BMBxs was not concordant, raising questions about using CTC gene expression to make clinical decisions.

Bone-induced c-kit expression in prostate cancer: a driver of intraosseous tumor growth.

Loss of BRCA2 function stimulates prostate cancer (PCa) cell invasion and is associated with more aggressive and metastatic tumors in PCa patients. Concurrently, the receptor tyrosine kinase c-kit is highly expressed in skeletal metastases of PCa patients and induced in PCa cells placed into the bone microenvironment in experimental models. However, the precise requirement of c-kit for intraosseous growth of PCa and its relation to BRCA2 expression remain unexplored. Here, we show that c-kit expression promotes migration and invasion of PCa cells. Alongside, we found that c-kit expression in PCa cells parallels BRCA2 downregulation. Gene rescue experiments with human BRCA2 transgene in c-kit-transfected PCa cells resulted in reduction of c-kit protein expression and migration and invasion, suggesting a functional significance of BRCA2 downregulation by c-kit. The inverse association between c-kit and BRCA2 gene expressions in PCa cells was confirmed using laser capture microdissection in experimental intraosseous tumors and bone metastases of PCa patients. Inhibition of bone-induced c-kit expression in PCa cells transduced with lentiviral short hairpin RNA reduced intraosseous tumor incidence and growth. Overall, our results provide evidence of a novel pathway that links bone-induced c-kit expression in PCa cells to BRCA2 downregulation and supports bone metastasis.

Safety and therapeutic effect of metronomic chemotherapy with cyclophosphamide and celecoxib in advanced breast cancer patients.

Metronomic chemotherapy (MCT), the chronic administration, at regular intervals, of low doses of chemotherapeutic drugs without extended rest periods, allows chronic treatment with therapeutic efficacy and low toxicity. Our preclinical results suggested that combined MCT with cyclophosphamide and celecoxib could inhibit breast cancer growth. The aim of this study was to determine the toxicity, safety and efficacy of oral MCT with cyclophosphamide 50 mg per orem daily and celecoxib 400 mg (200 mg per orem two-times a day) in advanced breast cancer patients. During the first stage of the study, the therapeutic response consisted of prolonged stable disease for ≥24 weeks in six out of 15 (40%) patients with a median duration of 37.5 weeks and a partial response in one out of 15 (response rate: 6.7%) patients lasting 6 weeks. The overall clinical benefit rate was 46.7%. The median time to progression was 14 weeks. Progression-free survival at 24 weeks was 40% and the 1-year overall survival rate was 46.7%. The adverse events were mild (gastric, grade 1; and hematologic, grade 1 or 2). No grade 3 or 4 toxicities were associated with the treatment. Evaluation of patients' quality of life showed no changes during the response period. MCT with cyclophosphamide plus celecoxib is safe and shows a therapeutic effect in advanced breast cancer patients.

[Immunomodulation and antiangiogenesis in cancer therapy. From basic to clinical research]. / Inmunomodulación y antiangiogénesis en la terapéutica oncológica. De la investigación básica a la clínica.

Basic and pre-clinic research in cellular and molecular oncology are the main supports accounting for the advancement in cancer therapeutics. The findings achieved, and their implementation in clinical practice are responsible for the permanent improvement in the treatment of the neoplastic disease. Our present objective is to summarize and discuss the pre-clinical findings in immunomodulation and anti-angiogenesis for the treatment of several types of tumors obtained in our Institute during the last 15 years, and the subsequent translation and application of the acquired experimental knowledge in a Phase I/II Clinical Trial. We present the results and mechanisms of action of antimetastatic immunomodulation with cyclophosphamide, the metronomic chemotherapy with different single drugs and their combinations, and finally the design and preliminary results of a clinical trial with metronomic chemotherapy for patients with advanced breast cancer.

The immune response and the therapeutic effect of metronomic chemotherapy with cyclophosphamide.

Metronomic chemotherapy (MCT) is a novel therapeutic strategy for cancer treatment endowed with an antiangiogenic effect. It refers to regular administration of low doses of cytotoxic drugs, with minimal or no drug-free breaks. Previously, we demonstrated the immunomodulating activity of a single low-dose of cyclophosphamide (Cy) and the antitumor effect of MCT with Cy on established rat lymphomas and sarcomas. Here, we examined whether the immune response is responsible for the antitumor effect of MCT with Cy on L-TACB lymphoma. Inbred e rats and nude mice were subcutaneously challenged with L-TACB. After 7 days, they were distributed into two experimental groups: 1) treated animals, which were injected IP with Cy (10 mg/kg body weight) three times per week, and 2) control animals, which received IP saline injections. Exponential growth and decay and tumor doubling time were calculated. Also, serum IL-10 levels were measured. One hundred percent of treated rats showed tumor regression versus 0% of control rats. The increase of tumor-induced IL-10 levels was reverted by the treatment with Cy. On the other hand, there were no tumor regressions, in treated or control nude mice. However, the tumor doubling times of treated nude mice were significantly higher than those of control mice, implying that other antitumor mechanism(s), independent of the adaptive immune response, might be taking place. Our present results indicate that modulation of the immune response would be involved in the antitumor effect of MCT with Cy, because the absence of the specific immune response impairs, at least in part, its therapeutic effect in a lymphoma tumor model.

Achievements and challenges in the use of metronomics for the treatment of breast cancer.

Two interesting therapeutic proposals for cancer treatment emerged at the beginning of the 21st century. The first one was metronomic chemotherapy, which refers to the chronic administration of chemotherapeutic agents, in low doses, without extended drug-free periods. Then, the idea of drug repositioning in oncology, the use of well-known drugs that were created for other uses to be utilized in oncology, gained strength. Shortly after, the two strategies were merged in one, named metronomics. Both approaches share several features which make metronomics an appealing choice for cancer treatment: use of known and approved drugs, thus diminishing the time necessary to enter to the clinic, therapeutic effect, low toxicity, oral administration, better life quality, low costs because of the use of, generally, out of patent drugs, possibility of use, even in countries with very low economic resources. Many chemotherapy and repurposed drugs were tested with metronomics approaches for the treatment of mammary cancer, the most common malignancy in women worldwide, leading to high rates of mortality. The wide range of therapeutic models studied, paralleled the wide range of responses obtained, like tumor growth and metastasis inhibition, overall survival increase, lack of toxicity, better life quality, among others. The accomplishments reached, and the challenges faced by researchers, are discussed.

Losartan improves the therapeutic effect of metronomic cyclophosphamide in triple negative mammary cancer models.

Metronomic chemotherapy refers to the minimum biologically effective doses of a chemotherapy agent given as a continuous regimen without extended rest periods. Drug repurposing is defined as the use of an already known drug for a new medical indication, different from the original one. In oncology the combination of these two therapeutic approaches is called "Metronomics". The aim of this work is to evaluate the therapeutic effect of cyclophosphamide in a metronomic schedule in combination with the repurposed drug losartan in two genetically different mice models of triple negative breast cancer. Our findings showed that adding losartan to metronomic cyclophosphamide significantly improved the therapeutic outcome. In both models the combined treatment increased the mice's survival without sings of toxicity. Moreover, we elucidated some of the mechanisms of action involved, which include a decrease of intratumor hypoxia, stimulation of the immune response and remodeling of the tumor microenvironment. The remarkable therapeutic effect, the lack of toxicity, the low cost of the drugs and its oral administration, strongly suggest its translation to the clinical setting in the near future.

Antitumor activity of new chemical compounds in triple negative mammary adenocarcinoma models.

AIM: According to the need for the development of new anticancer agents, we have synthetized novel bioactive compounds and aimed to determine their antitumor action. MATERIALS & METHODS: We describe in vitro studies evaluating the effect of 35 novel chemical compounds on two triple negative murine mammary adenocarcinoma tumors. RESULTS & CONCLUSION: Three compounds were selected because of their high antitumor activity and their low toxicity to normal cells. Their effect on tumor cells apoptosis, clonogenicity and migratory capacity, were determined. We found that the selected compounds showed inhibition of viability and clonogenic capacity, and promotion of apoptosis. They also decreased the migratory capacity of tumor cells. The results obtained suggest the likelihood of their future use as antitumor and/or antimetastatic agents.

Highlights from the 1st Latin American meeting on metronomic chemotherapy and drug repositioning in oncology, 27-28 May, 2016, Rosario, Argentina.

Following previous metronomic meetings in Marseille (2011), Milano (2014), and Mumbai (2016), the first Latin American metronomic meeting was held in the School of Medical Sciences, National University of Rosario, Rosario, Argentina on 27 and 28 of May, 2016. For the first time, clinicians and researchers with experience in the field of metronomics, coming from different countries in Latin America, had the opportunity of presenting and discussing their work. The talks were organised in three main sessions related to experience in the pre-clinical, and clinical (paediatric and adult) areas. The different presentations demonstrated that the fields of metronomic chemotherapy and repurposing drugs in oncology, known as metronomics, constitute a branch of cancer therapy in permanent evolution, which have strong groups working in Latin America, both in the preclinical and the clinical settings including large, adequately designed randomised studies. It was shown that metronomics offers treatments, which, whether they are combined or not with the standard therapeutic approaches, are not only effective but also minimally toxic, with the consequent improvement of the patient's quality of life, and inexpensive, a feature very important in low resource clinical settings. The potential use of metronomic chemotherapy was proposed as a cost/effective treatment in low-/middle-income countries, for adjuvant therapy in selected tumours. The fundamental role of the governmental agencies and non-governmental alliances, as the Metronomic Global Health Initiative, in supporting this research with public interest was underlined.

Antitumoral and antimetastatic effects of metronomic chemotherapy with cyclophosphamide combined with celecoxib on murine mammary adenocarcinomas.

PURPOSE: Metronomic chemotherapy (MCT) refers to the chronic and equally spaced administration of low doses of different chemotherapy drugs, without extended interruptions. Previously, we demonstrated the antitumor effect of MCT with cyclophosphamide (Cy) in a mouse mammary adenocarcinoma model. Herein, we investigated the therapeutic efficacy of metronomic Cy combined with celecoxib (Cel) in two murine mammary adenocarcinoma models. METHODS: Mice were s.c. challenged with M-234 p or M-406 mammary tumors and from day 5 or 8 on, respectively, treated with: (I) no treatment (controls); (II) Cy in the drinking water (25-30 mg/kg body weight/day); (III) Cel (30 mg/kg p.o.), five times/week; (IV) treated as II + III. Mice challenged i.v. with M-234 p or M-406 tumor cells received, on day 3, the same treatments. RESULTS: We found that MCT with Cy plus Cel inhibited tumor growth decreased lung metastases, and increased the median survival time, in both tumor models, having very low toxicity. MCT with Cy combined with Cel was more effective than each monotherapy. CONCLUSIONS: The therapeutic benefits of combined MCT with cyclophosphamide plus celecoxib on mammary adenocarcinomas together with its very low toxicity profile warrant further study in an attempt to make the translation into the clinic.

Inmunomodulación y antiangiogénesis en la terapéutica oncológica: De la investigación básica a la clínica / Immunomodulation and antiangiogenesis in cancer therapy: From basic to clinical research

La investigación básica y pre-clínica en oncología celular y molecular son pilares fundamentales en los que se apoyan la mayoría de los adelantos en la terapéutica del cáncer. Los hallazgos obtenidos y su aplicación en la práctica clínica constituyen la causa del avance sostenido en el tratamiento de la enfermedad neoplásica. El objetivo de este trabajo es resumir y discutir los resultados pre-clínicos en inmunomodulación y anti-angiogénesis para el tratamiento de diversos tipos de tumores, obtenidos en nuestro Instituto durante los últimos 15 años, y la posterior traslación y aplicación del conocimiento experimental en un Ensayo Clínico Fase I/II. Se describen los resultados que contribuyeron a descifrar los mecanismos de acción de la inmunomodulación antimetastásica con ciclofosfamida, la quimioterapia metronómica con diferentes drogas únicas o combinaciones, y finalmente el diseño y resultados preliminares de un ensayo clínico de quimioterapia metronómica para pacientes con cáncer de mama avanzado.

Basic and pre-clinic research in cellular and molecular oncology are the main supports accounting for the advancement in cancer therapeutics. The findings achieved, and their implementation in clinical practice are responsible for the permanent improvement in the treatment of the neoplastic disease. Our present objective is to summarize and discuss the pre-clinical findings in immunomodulation and anti-angiogenesis for the treatment of several types of tumors obtained in our Institute during the last 15 years, and the subsequent translation and application of the acquired experimental knowledge in a Phase I/II Clinical Trial. We present the results and mechanisms of action of antimetastatic immunomodulation with cyclophosphamide, the metronomic chemotherapy with different single drugs and their combinations, and finally the design and preliminary results of a clinical trial with metronomic chemotherapy for patients with advanced breast cancer.