BackgroundWhile it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment.Methodology and Principal FindingsWe examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2–4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patient's breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patient's original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from the organ culture, and abrogated xenograft tumor formation.ConclusionsCytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.

Disulfiram (DSF) is an aldehyde dehydrogenase inhibitor currently used for the treatment of alcoholism. Here, we show that multiple myeloma (MM) cell lines and primary cells from newly diagnosed and relapsed/resistant patients affected by MM, acute myeloid and lymphoblastic leukemia are significantly sensitive to DSF alone and in combination with copper. These effects are present at doses lower than those achievable in vivo after DSF standard administration. The cytotoxic effect achieved by this treatment is comparable to that obtained by conventional chemotherapy and is absent in normal hematopoietic cells. In addition, we found that DSF plus copper induces loss of mitochondrial membrane potential, triggers reactive oxygen species (ROS) production and activates executioner caspases. DSF-copper-induced apoptosis and caspases activation are strongly reversed by antioxidant N-acetylcysteine, thus indicating a critical role of ROS. These results might suggest the use of the old drug DSF, alone or in combination with copper, in the treatment of hematological malignancies.The commonly used alcohol-abuse deterrent disulfiram (DSF) is a member of the dithiocarbamate family, a broad class of molecules with the ability to complex metals, such as copper, and react with sulfhydryl groups and glutathione (GSH). DSF has been used for decades in the treatment of alcoholism for its ability to inhibit irreversibly the aldehyde dehydrogenase.1,2 Several studies from 1970s (e.g., Lewison 1977) 3 have shown that DSF and its metabolites can enhance the effect of some chemotherapeutics, and it is considered a convincing anticancer drug both in vivo and in human patients. The case report described by Lewison was the first on DSF activity against breast cancer.3 Then the compound or its main metabolite was successfully used in a phase II clinical trial with 64 high-risk breast cancer patients and in hepatic metastases and in a patient with stage IV metastatic ocular melanoma. 4 More recently, there are ongoing clinical trials for DSF (Antabuse) as an adjuvant therapy (ClinicalTrials.gov Identifier NCT00312819) or combined with copper gluconate (ClinicalTrials.gov Identifier NCT00742911) against advanced solid cancers. However, a clear explanation for the antitumor effect of DSF is still missing. 2,4It has been shown that association of DSF with copper forms a complex with potent proteasome inhibiting and apoptosis-inducing activity on several tumors, both in vitro and in vivo. 1,2,4-6Moreover, DSF has also been found to reverse the resistance of human tumors to chemotherapeutic drugs by blocking maturation of the P-glycoprotein membrane pump; inhibit activation of nuclear factor-kB; decrease angiogenesis, tumor growth in vivo, matrix metalloproteinases activity and cancer cell invasiveness. 4,5 The proapoptotic activity of DSF has been attributed to redox-related mitochondrial membrane permeabilization, followed by complexation with zinc/copper and inhibition of Zn-dependent matrix metalloproteinases or Cu/Zn superoxide dismutase. The...

The combination of proteasome inhibitors and TRAIL synergizes to induce the destruction of chemoresistant neoplastic thyrocytes and could represent a promising therapeutic strategy for the treatment of anaplastic thyroid carcinoma.

Abstract. Thyroid carcinoma is the most common endocrine neoplasm, with the highest mortality rate of all the endocrine cancers. Among the endocrine malignancies, ~80% are papillary thyroid carcinomas (PTCs). In the initiation and progression of this tumor, genetic alterations in the mitogen-associated protein kinase pathway, including RAS point mutations, RET/PTC oncogene rearrangements and BRAF point mutations, play an important role, particularly in deciding targeted therapy. In the present study, a small population of thyroid tumor cells, known as tumor spheres, were isolated and characterized from PTC surgical samples. These spheres can be expanded indefinitely in vitro and give rise to differentiated adherent cells when cultivated in differentiative conditions. The present study showed by reverse transcription-polymerase chain reaction and flow cytometric analysis that the undifferentiated PTC cells exhibited a characteristic antigen expression profile of adult progenitor/stem cells. The cells were more resistant to chemotherapeutics, including bortezomib, taxol, cisplatin, etoposide, doxorubicin and vincristine, than differentiated PTC cells and the majority possessed a quiescent status, as revealed by the various cell cycle characteristics and anti-apoptotic protein expression. Such advances in cancer thyroid stem cell biology may provide relevant information for future targeted therapies.

Background: PTX is one of the most widely used drug in oncology due to its high efficacy against solid tumors and several hematological cancers. PTX is administered in a formulation containing 1:1 Cremophor ® EL (polyethoxylated castor oil) and ethanol, often responsible for toxic effects. Its encapsulation in colloidal delivery systems would gain an improved targeting to cancer cells, reducing the dose and frequency of administration.

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Acute myeloid leukemia (AML) is a malignant disease characterized by abnormal proliferation of clonal precursor cells. Although different strategies have been adopted to obtain complete remission, the disease actually progresses in about 60–70% of patients. Bortezomib has been used in multiple myeloma and other lymphoid malignancies because of its antitumor activity. Here we examined the sensitivity of bone marrow cells from AML patients (34 patients: 25 newly diagnosed, 4 relapsed, 5 refractory) to bortezomib alone or in combination with TRAIL, a member of the TNF family that induces apoptosis in tumor cells while sparing normal cells. Bortezomib induced cell death in blasts from each patient sample. The cytotoxic effect was dose- and time-dependent (concentration from 0.001 to 10 µM for 24 and 48 h) and was associated with a downregulation of Bcl-xL and Mcl-1, an upregulation of TRAIL-R1, TRAIL-R2, p21, activation of executioner caspases and a loss of the mitochondrial membrane potential. Moreover, low doses of bortezomib primed TRAIL-resistant AML cells for enhanced TRAIL-mediated killing. These results suggest that a combination of proteasome inhibitors and TRAIL could be effective for treating AML patients, even patients who are refractory to conventional chemotherapy.