Gabriel L. Fiszman

Molecular Mechanisms of Trastuzumab Resistance in HER2 Overexpressing Breast Cancer

The epidermal growth factor receptor 2 (HER2) is a tyrosine kinase overexpressed in nearly 20% to 25% of invasive breast cancers. Trastuzumab is a humanized monoclonal antibody that targets HER2. The majority of patients with metastatic breast cancer initially respond to trastuzumab, however, within 1 year of treatment disease progresses. Several molecular mechanisms have been described as contributing to the development of trastuzumab resistance. They could be grouped as impaired access of trastuzumab to HER2, upregulation of HER2 downstream signaling pathways, signaling of alternative pathways, and impaired immune antitumor mechanisms. However, since many of them have overlapping effects, it would be of great clinical impact to identify the principal signaling pathways involved in drug resistance. Significant efforts are being applied to find other therapeutic modalities besides trastuzumab treatment to be used alone or in combination with current modalities.

Activation of muscarinic cholinergic receptors induces MCF-7 cells proliferation and angiogenesis by stimulating nitric oxide synthase activity

Muscarinic acetylcholine receptors (mAChR) are members of the G-protein coupled receptor family. These receptors play key physiological roles and changes in their expression and/or function are involved in several diseases. We had previously demonstrated that mAChR expression is up regulated in three different cell lines derived from distinct murine mammary adenocarcinomas that spontaneously arose in BALB/c female mice, in comparison with normal murine mammary cells. Stimulation of mAChR with the muscarinic agonist carbachol (CARB) potentiated different steps of tumor progression. We here evidence that similarly to previous results obtained in mice, human breast tumor homogenates over expressed mAChR in comparison with normal breast tissue. Thus, to test the muscarinic actions on human breast adenocarcinoma cells we investigate the effect of CARB on MCF-7 cells proliferation and neovascular response. Particularly we observe that: CARB stimulates tumor cells proliferation, being 10-9 M the maximal effective dose for the muscarinic agonist. This action was due to M3 and M1 receptors activation being nitric oxide synthase (NOS) its effector enzyme via phospholipase C and protein kinase C signaling pathway. NOS1 and NOS3 isoforms are expressed in MCF-7 cells and its activation by CARB triggers nitric oxide synthesis and vascular endothelial growth factor expression increasing blood vessels formation induced by mammary tumor cells in vivo. We can conclude that non-neuronal cholinergic system activation stimulates MCF-7 tumor cells growth and neovascular response promoting tumor progression.

Suicide gene and cytokines combined nonviral gene therapy for spontaneous canine melanoma.

Canine spontaneous melanoma is a highly aggressive tumor resistant to current therapies. We evaluated the safety, efficacy and antitumor effects of direct intratumor injections of lipoplexes encoding herpes simplex thymidine kinase coadministrated with ganciclovir, and irradiated transgenic xenogeneic cells secreting 20–30 μg day−1 of human granulocyte–macrophage colony-stimulating factor and interleukin-2. Toxicity was minimal or absent in all patients. This combined treatment (CT) induced tumor regression and a pronounced immune cell infiltration. The objective responses (47%: 21/45) averaged 80% of tumor mass loss. Local CT also induced systemic antitumor response evidenced by complete remission of one pulmonary metastasis and by the significantly higher percentage of metastasis-free patients (76: 34/45)) until the study ending compared to untreated (UC: 29%, 5/17), surgery-treated (CX: 48%, 11/23) or suicide gene-treated controls (SG: 56%, 9/16) (Fishers exact test). CT significantly improved median survival time: 160 (57–509) days compared to UC (69 (10–169)), CX (82 (43–216)) or SG (94 (46–159)). CT also increased (P<0.00001, Kaplan–Meier analysis) metastasis-free survival: >509 (57–509) days with respect to UC: 41 (10–169), CX: 133 (43–216) and SG: >159 (41–159). Therefore, CT controlled tumor growth by delaying or preventing distant metastasis, thereby significantly extending survival and recovering the quality of life.

Establishment of an in vitro estrogen-dependent mouse mammary tumor model: a new tool to understand estrogen responsiveness and development of tamoxifen resistance in the context of stromal–epithelial interactions

Currently, to our knowledge, there are no continuous cell lines derived from estrogen dependent, tamoxifen sensitive spontaneous mouse mammary carcinomas. We describe here the establishment and characterization of a cell line derived from the M05 mouse mammary tumor, LM05-Mix, composed of both an epithelial and a fibroblastic component. From it the respective epithelial LM05-E and fibroblastic LM05-F cell lines were generated by limiting dilution. Immunofluorescence studies confirmed that the epithelial cells were positive for E-cadherin, cytokeratins and vimentin whereas the fibroblastic cells were negative for the epithelial markers and positive for α-smooth muscle actin and vimentin. Both cell types expressed estrogen and progesterone receptors, although only the epithelial LM05-E cells were stimulated by estradiol and inhibited by tamoxifen. In the bicellular LM05-Mix cell line estradiol proved to stimulate cell proliferation whereas the response to tamoxifen was dependent on confluency and the degree of epithelial-fibroblastic interactions. The presence of membrane estrogen receptors in both cell types was suggested by the achievement of non-genomic responses to short treatments with estradiol, leading to the phosphorylation of ERK1/2. Finally, cytogenetic studies suggest that these two cell types represent independent cell populations within the tumor and would not be the result of an epithelial-mesenchymal transition. This model presents itself as a valuable alternative for the study of estrogen responsiveness and tamoxifen resistance in the context of epithelial-stromal interactions.

Herpes simplex virus thymidine kinase/ganciclovir system in multicellular tumor spheroids

We have developed multicellular spheroids (MCS) established from LM05e and LM3 spontaneous Balb/c-murine mammary adenocarcinoma and B16 C57-murine melanoma derived cell lines as an in vitro model to study the efficacy of the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide system. We demonstrated for the first time that HSVtk-expressing cells assembled as MCS manifested a GCV resistance phenotype compared to the same cells grown as sparse monolayers. HSVtk-expressing LM05e, LM3 and B16 spheroids were 16-, three- and nine-fold less sensitive to GCV than their respective monolayers, even though they could express transgenes 10-, eight- and five-fold more efficiently. Mixed populations of HSVtk− and their respective βgal-expressing cells displayed a cell-type specific bystander effect that was higher in monolayers than in MCS. However, HSVtk−expressing cells in two- or three-dimensional cultures were always significantly more sensitive to GCV than the βgal-expressing counterparts, supporting the feasibility of this suicide approach in vivo. We present evidence showing that HSVtk-expressing tumor cells, when transferred from monolayers to MCS, displayed: (i) lower GCV cytotoxic activity and bystander effect; (ii) higher and efficient expression of genes transferred as lipoplexes; (iii) lower cell proliferation rates; and (iv) changes in intracellular Bax/Bcl-xL rheostat of mitochondria-mediated apoptosis.

Functional changes in murine mammary cancer cells elicited by CoCl2-induced hypoxia

Low O(2) levels in solid tumors are associated with increase in hypoxia-inducible factor 1alpha (HIF-1alpha). The present study examines functional changes involved in adaptation to hypoxia of the LMM3 mammary tumor cell line, using CoCl(2) as hypoxic mimetic. Our results showed that LMM3 cells were not only tolerant to 150 microM CoCl(2) but they can overgrowth in vitro respect to untreated cells. Hypoxia inhibited cell invasion, migration, MMP-9 activity and NO levels. Macrophage cytotoxicity augmented under hypoxia but was blunted by conditioned media from tumor cells. In vivo tumorigenicity of CoCl(2)-treated cells was greater than controls. Our results show stabilization of HIF-1alpha in LMM3 cells under CoCl(2) and functional changes associated with enhanced cell survival and growth but not with tumor dissemination.

A laboratory scale device for microencapsulation of genetically engineered cells into alginate beads

The microencapsulation of recombinant cells, widely used for in vitro high-density cell culture, is a novel and potentially cost-effective method of in vivo heterologous protein delivery, where the protein producing cells are immunologically protected from tissue rejection. We report here a simple, reliable and inexpensive laboratory method to generate calcium alginate microcapsules containing genetically engineered, interleukin-2 expressing, Chinese hamster ovary (CHO) cells.

PKCδ Inhibition Impairs Mammary Cancer Proliferative Capacity But Selects Cancer Stem Cells, Involving Autophagy

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate diverse cellular functions including cell death, proliferation, and survival. Recent studies have reported that PKCδ, are involved in apoptosis or autophagy induction. In the present study we focused on how PKCδ regulates proliferation and cancer stem cell (CSC) properties of the hormone‐independent mammary cancer cell line LM38‐LP, using pharmacological and genetic approaches. We found that pharmacological inhibition of PKCδ, by Rottlerin treatment, impairs in vitro LM38‐LP proliferation through cell cycle arrest, inducing the formation of cytoplasmic‐vacuoles. Using immunofluorescence we confirmed that Rottlerin treatment induced the apparition of LC3 dots in cell cytoplasm, and increased autophagy flux. On the other side, the same treatment increased CSC growth rate and self‐renewal. Furthermore, Rottlerin pre‐treatment induced in CSC the development of a “grape‐like” morphology when they are growing in 3D cultures (Matrigel), usually associated with a malignant phenotype, as well as an increase in the number of experimental lung metastasis when these cells were inoculated in vivo. The PKCδ knockdown, by RNA interference, induced autophagy and increased CSC number, indicating that these effects are indeed exerted through a PKCδ dependent pathway. Finally, the increase in the number of mammospheres could be reversed by a 3MA treatment, suggesting that autophagy mechanism is necessary for the increased of CSC self‐renewal induced by PKCδ inhibition. Here we demonstrated that PKCδ activity exerts a dual role through the autophagy mechanism, decreasing proliferative capacity of mammary tumor cells but also regulating tumor stem cell self‐renewal. J. Cell. Biochem. 117: 730–740, 2016.

Autophagy Protects from Trastuzumab-Induced Cytotoxicity in HER2 Overexpressing Breast Tumor Spheroids.

Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance.

Immunoglobulin G from Breast Cancer Patients Regulates MCF-7 Cells Migration and MMP-9 Activity by Stimulating Muscarinic Acetylcholine Receptors

PurposeWe have previously reported the expression of muscarinic acetylcholine receptors (mAChR) in human breast tumors. The activation of these receptors triggered tumor cell proliferation. Considering that invasion and metastasis is the major cause of death in cancer, we investigated the action of autoantibodies against mAChR derived from breast cancer patients in stage I (T1N0Mx-IgG) on MCF-7 cells migration and metalloproteinase-9 (MMP-9) activity. We also analyzed the participation of phospholipase C/nitric oxide synthase/protein kinase C pathway.MethodsImmunoglobulin G (IgG) was purified by chromatography in protein G-agarose from blood samples of breast cancer patients obtained under informed consent. Migration was assayed by an in vitro wound assay. MMP-9 activity was quantified by zymography.ResultsT1N0Mx-IgG promoted tumor cell migration and increased MMP9 activity mimicking the action of the muscarinic agonist carbachol. This effect was reduced not only by the presence of atropine but also by 4-DAMP or tropicamide, antagonists for M3 and M4 mAChR subtypes respectively. The actions of T1N0Mx-IgG and carbachol on MCF-7 cells, involved the participation of phospholipase C/nitric oxide synthase/protein kinase C pathway.ConclusionsIgG from breast cancer patients in stage I could be promoting tumor progression by regulating migration and MMP-9 activity in tumor cells via mAChR activation. The presence of these autoantibodies could be determining the prognosis of breast cancer in these patients.