María I. Díaz Bessone

Autophagy: Friend or Foe in Breast Cancer Development, Progression, and Treatment

Autophagy is a catabolic process responsible for the degradation and recycling of long-lived proteins and organelles by lysosomes. This degradative pathway sustains cell survival during nutrient deprivation, but in some circumstances, autophagy leads to cell death. Thereby, autophagy can serve as tumor suppressor, as the reduction in autophagic capacity causes malignant transformation and spontaneous tumors. On the other hand, this process also functions as a protective cell-survival mechanism against environmental stress causing resistance to antineoplastic therapies. Although autophagy inhibition, combined with anticancer agents, could be therapeutically beneficial in some cases, autophagy induction by itself could lead to cell death in some apoptosis-resistant cancers, indicating that autophagy induction may also be used as a therapy. This paper summarizes the most important findings described in the literature about autophagy and also discusses the importance of this process in clinical settings.

Involvement of PKC delta (PKCδ) in the resistance against different doxorubicin analogs

Doxorubicin is an anti-tumor antibiotic widely used in the management of cancer patients. Its main mechanism of action involves the generation of DNA damage and the inhibition of topoisomerase II, promoting apoptosis. AD 198 is a novel doxorubicin analog devoid of DNA binding and topoisomerase II inhibitory capacities. It has been proposed that AD 198 induces apoptosis by activating protein kinase C delta (PKCδ); a PKC isoform described as growth inhibitory in a large number of cell types. We have previously demonstrated that PKCδ overexpression in NMuMG cells induced the opposite effect, promoting proliferation and cell survival. In this study, we found that PKCδ overexpression confers an enhanced cell death resistance against AD 198 cytotoxic effect and against AD 288, another doxorubicin analog that preserves its mechanism of action. These resistances involve PKCδ-mediated activation of two well-known survival pathways: Akt and NF-κB. While the resistance against AD 198 could be abrogated upon the inhibition of either Akt or NF-κB pathways, only NF-κB inhibition could revert the resistance to AD 288. Altogether, our results indicate that PKCδ increases cell death resistance against different apoptosis inductors, independently of their mechanism of action, through a differential modulation of Akt and NF-κB pathways. Our study contributes to a better understanding of the mechanisms involved in PKCδ-induced resistance and may greatly impact in the rationale design of isozyme-specific PKC modulators as therapeutic agents.

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.

Myoepithelial and luminal breast cancer cells exhibit different responses to all-trans retinoic acid

PurposeBreast cancer is the leading cause of death among women worldwide. The exact role of luminal epithelial (LEP) and myoephitelial (MEP) cells in breast cancer development is as yet unclear, as also how retinoids may affect their behaviour. Here, we set out to evaluate whether retinoids may differentially regulate cell type-specific processes associated with breast cancer development using the bi-cellular LM38-LP murine mammary adenocarcinoma cell line as a model.Materials and methodsThe bi-cellular LM38-LP murine mammary cell line was used as a model throughout all experiments. LEP and MEP subpopulations were separated using inmunobeads, and the expression of genes known to be involved in epithelial to mysenchymal transition (EMT) was assessed by qPCR after all-trans retinoic acid (ATRA) treatment. In vitro invasive capacities of LM38-LP cells were evaluated using 3D Matrigel cultures in conjunction with confocal microscopy. Also, in vitro proliferation, senescence and apoptosis characteristics were evaluated in the LEP and MEP subpopulations after ATRA treatment, as well as the effects of ATRA treatment on the clonogenic, adhesive and invasive capacities of these cells. Mammosphere assays were performed to detect stem cell subpopulations. Finally, the orthotopic growth and metastatic abilities of LM38-LP monolayer and mammosphere-derived cells were evaluated in vivo.ResultsWe found that ATRA treatment modulates a set of genes related to EMT, resulting in distinct gene expression signatures for the LEP or MEP subpopulations. We found that the MEP subpopulation responds to ATRA by increasing its adhesion to extracellular matrix (ECM) components and by reducing its invasive capacity. We also found that ATRA induces apoptosis in LEP cells, whereas the MEP compartment responded with senescence. In addition, we found that ATRA treatment results in smaller and more organized LM38-LP colonies in Matrigel. Finally, we identified a third subpopulation within the LM38-LP cell line with stem/progenitor cell characteristics, exhibiting a partial resistance to ATRA.ConclusionsOur results show that the luminal epithelial (LEP) and myoephitelial (MEP) mammary LM38-P subpopulations respond differently to ATRA, i.e., the LEP subpopulation responds with increased cell cycle arrest and apoptosis and the MEP subpopulation responds with increased senescence and adhesion, thereby decreasing its invasive capacity. Finally, we identified a third subpopulation with stem/progenitor cell characteristics within the LM38-LP mammary adenocarcinoma cell line, which appears to be non-responsive to ATRA.

Involvement of protein kinase C α and δ activities on the induction of the retinoic acid system in mammary cancer cells

It has been established that retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through the interaction with different members of the protein kinase C (PKC) family. Till nowadays the nature and extension of this interaction is not well understood. Due to the cytostatic and differentiating effects of retinoids, in the present study we propose to evaluate whether the crosstalk between the retinoid system and the PKC pathway could become a possible target for breast cancer treatment. We could determine that ATRA (all‐trans retinoic) treatment showed a significant growth inhibition due to (G1 or G2) cell cycle arrest both in LM3 and SKBR3, a murine and human mammary cell line respectively. ATRA also induced a remarkable increase in PKCα and PKCδ expression and activity. Interestingly, the pharmacological inhibition of these two PKC isoforms prevented the activation of retinoic acid receptors (RARs) by ATRA, indicating that both PKC isoforms are required for RARs activation. Moreover, PKCδ inhibition also impaired ATRA‐induced RARα translocation to the nucleus. In vivo assays revealed that a combined treatment using ATRA and PKCα inhibitors prevented lung metastatic dissemination in an additive way. Our results clearly indicate that ATRA modulates the expression and activity of different PKCs. Besides inducing cell arrest, the activity of both PKC is necessary for the induction of the retinoic acid system. The combined ATRA and PKCα inhibitors could be an option for the hormone‐independent breast cancer treatment.

The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines

BackgroundLung cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths worldwide. Up to 80% of cancer patients are classified as non-small-cell lung cancer (NSCLC) and cisplatin remains as the gold standard chemotherapy treatment, despite its limited efficacy due to both intrinsic and acquired resistance. The CK2 is a Ser/Thr kinase overexpressed in various types of cancer, including lung cancer. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to CK2 substrates thus preventing the enzyme activity. The aim of this work was to analyze the effects of CIGB-300 treatment targeting CK2-dependent signaling pathways in NSCLC cell lines and whether it may help improve current chemotherapy treatment.MethodsThe human NSCLC cell lines NCI-H125 and NIH-A549 were used. Tumor spheroids were obtained through the hanging-drop method. A cisplatin resistant A549 cell line was obtained by chronic administration of cisplatin. Cell viability, apoptosis, immunoblotting, immunofluorescence and luciferase reporter assays were used to assess CIGB-300 effects. A luminescent assay was used to monitor proteasome activity.ResultsWe demonstrated that CIGB-300 induces an anti-proliferative response both in monolayer- and three-dimensional NSCLC models, presenting rapid and complete peptide uptake. This effect was accompanied by the inhibition of the CK2-dependent canonical NF-κB pathway, evidenced by reduced RelA/p65 nuclear levels and NF-κB protein targets modulation in both lung cancer cell lines, as well as conditionally reduced NF-κB transcriptional activity. In addition, NF-κB modulation was associated with enhanced proteasome activity, possibly through its α7/C8 subunit. Neither the peptide nor a classical CK2 inhibitor affected cytoplasmic β-CATENIN basal levels. Given that NF-κB activation has been linked to cisplatin-induced resistance, we explored whether CIGB-300 could bring additional therapeutic benefits to the standard cisplatin treatment. We established a resistant cell line that showed higher p65 nuclear levels after cisplatin treatment as compared with the parental cell line. Remarkably, the cisplatin-resistant cell line became more sensitive to CIGB-300 treatment.ConclusionsOur data provide new insights into CIGB-300 mechanism of action and suggest clinical potential on current NSCLC therapy.

Mammary Gland Organoids

The study of the mechanisms that regulate development and tumorigenesis is a complex undertaking that requires a variety of model systems to test hypothesis that embrace all levels of organization: from single cells to organs. In the mammary gland field, the use of three-dimensional culture systems has provided a platform to study, in a physiologically relevant setting, cell biology in context. In the late 50’s methods to isolate primary mammary organoids were established and since then they have been increasingly used to understand cell behavior. In this chapter we embrace, in a historical perspective, the key findings carried out using primary mammary organoids considering that the broadening of our knowledge will, in the future, rely increasingly on this kind of tridimensional culture setting.

Abstract 965: Two in one: nanotechnology based strategies for the treatment of ER+ breast cancer

Understanding endocrine resistance mechanisms is key to develop new therapeutic strategies. We focus on the role the tumor microenvironment plays as a modulator of endocrine therapy resistance in breast cancer, in particular associated to Tamoxifen. Studies show that the use of nanoparticles (NPs) as antitumor drug delivery systems is a good strategy to improve the efficacy and decrease secondary effects of conventional chemotherapies. In this context we hypothesize that a therapeutic strategy based on the use of Tamoxifen carried in NPs coated with the tumor penetrating peptide iRGD, would be more effective than conventional Tamoxifen. Neuropilin-1, that mediates iRGD induced endocytosis, has been shown to be associated to breast cancer stem cells. Thus, we postulate that our multifunctional NPs would be effective in reducing this cell population, contrary to what is observed with free Tamoxifen. Moreover, iRGD blocks the interaction between integrin β1 and fibronectin, a mechanism we have previously shown induces Tamoxifen resistance (Pontiggia et al. 2012). NPs were synthesized with polyethyleneglycol and polycaprolactone and coated with the iRGD peptide. This peptide was constructed with a FAM fluorophore in order to track the NPs. To evaluate the cell uptake, MCF-7 cells were incubated for 5 h with iRGD-NP or FAM-NP as a control. NP entry was higher when the NPs were coated with iRGD, both in 2D and in 3D cultures. Cell viability experiments revealed that Tamoxifen encapsulated in NPs was more effective than the free drug (p Citation Format: Maria Ines Diaz Bessone, Lorena Simon-Gracia, Pablo Scodeller, Tambet Teesalu, Marina Simian. Two in one: nanotechnology based strategies for the treatment of ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 965. doi:10.1158/1538-7445.AM2017-965

Abstract 1289: The synthetic peptide CIGB-300 inhibits nuclear factor κB (NF-κB) affecting the survival and chemoresistance of human lung cancer cells

Lung cancer is the leading cause of cancer deaths worldwide and despite significant progress, current therapies are limited in efficacy. The CK2 Ser/Thr kinase has been historically linked with cancer. It is involved in cell proliferation, survival and apoptosis by modulating diverse signaling pathways, including Wnt and NF-κB among the most relevant. CIGB-300 is an antitumor peptide with a novel mechanism of action, capable of binding to CK2 substrates thus preventing the enzyme activity. Previously, we have determined that CIGB-300 induces apoptosis through caspase-3 activation in different lung cancer cell lines. Moreover, CIGB-300 strongly inhibited RelA/NF-κB (p65) nuclear translocation, even in the presence of a phorbol-ester activating stimulus. NF-κB activation is known to reduce chemotherapy efficiency in different malignancies, including lung cancer. Based on this evidence, we hypothesize that supplementing cisplatin with CIGB-300 would improve the treatment efficiency. Indeed, we observed by Western blot that nuclear p65 levels were highly increased after treating human NCI-H125 cells with cisplatin. Moreover, when cells were treated with cisplatin plus CIGB-300, NF-κB activation was completely abolished. Therefore, the CIGB-300 effect on NF-κB signaling pathway prevails over cisplatin. These promising results on NF-κB inhibition led us to evaluate the combined treatment in chemoresistant setting. For this purpose we developed a cisplatin resistant A549 lung cancer cell line (A549-Rcisp) by the chronic administration of cisplatin during six months. A549-Rcisp viability was 40% higher than parental cells, confirming the cisplatin-acquired resistance. Remarkably, cisplatin resistant cells showed a significant increase in CIGB-300 sensitivity as compared to the parental cell line (p Given that NF-κB dimer stability is regulated by the proteasome-selective proteolysis of its inhibitory proteins, we studied the effect of CIGB-300 on this process. Surprisingly, we observed a significant increase on protease activities associated with the proteasome after 30 minutes of CIGB-300 treatment. Thus, proteasome complex is a newly identified target of CIGB-300 that could be relevant for its mechanism of action and deserves further exploration in order to determine the association with the observed perturbation of different signaling pathways. Citation Format: Stefano M. Cirigliano, Maria Ines Diaz Bessone, Carolina Flumian, Damian E. Berardi, Silvio Perea, Elisa Bal De Kier Joffe, Hernan Farina, Laura Todaro, Alejandro Urtreger. The synthetic peptide CIGB-300 inhibits nuclear factor κB (NF-κB) affecting the survival and chemoresistance of human lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1289.

Abstract 5203: Differential response to retinoid treatment in mouse and human mammary tumor cell lines with alterations in protein kinase C (PKC) expression

PKC is a serine-threonine kinase family that controls malignant transformation and metastatic dissemination. ATRA is the main active metabolite of vitamin A. Some evidences indicate that PKCδ may regulate the expression of some retinoid acid (RA) dependent genes, and others indicate that retinoids could alter PKCα intracellular localization; both processes would lead to cell differentiation. In this work we have developed human (MDA-MB 231) and murine (LM3) cell models overexpressing PKCα or PKCδ, in order to determine whether PKC expression alters the sensitivity to retinoids treatment (ATRA). The effect of ATRA was studied in vitro, analyzing biological responses related to tumor growth. In LM3 cells, only PKCα overexpression was able to reduce in vitro population doubling time (PDT) as compared to control (PDT: 14,8±2,3 h vs 21,2±3,1 h for LM3-PKCα y LM3-Vector respectively). Moreover, these cells also responded to retinoid treatment with a significant delay in cell proliferation (PDT: 24,3±4,2 h vs. 14,8±2,3 h for LM3-PKCα ATRA treated or not respectively. In MDA-MB 231 derived cell lines, PKC overexpression as well as ATRA treatment have no effect on proliferative potential. No differences were observed on migratory and invasive capabilities either. Interestingly, in LM3, PKCδ overexpression induced an important increase in proteolytic enzymes secretion, which correlates with its major invasiveness, but this increase had no impact on in vivo metastatic dissemination. Only PKCα overexpression increased this parameter (lung nodes, median (range): 55 (20-75) vs 0 (0-10) for LM3-PKCα y LM3-Vector respectively). Contrary to LM3-PKCδ in MDA-PKCδ cells we could detect a decrease on proteolytic enzymes production and invasive capability. Moreover, colonies growing in Matrigel as 3D cultures showed a small and branched structures. Finally we studied whether the overexpression of α and δ PKC isoforms is able to alter the activity of retinoic acid responsive elements (RARE) through a reporter gene assay. On LM3 model, the constitutive expression of PKCδ highly increased RARE dependent activity. Surprisingly, in MDA-MB231 derived sublines, we could detect a significant increase on RARE activity when cells were treated with ATRA, Altogether, these results suggest that PKCα overexpression confers a more aggressive phenotype in LM3 model, but also makes these cells sensitive to ATRA effects. We could hypothesize, that the absence of response to ATRA treatment displayed by MDA-MB 231 sublines can be explained by their lack of RARβ, which is implied in AP-1 transrepression in response to ATRA. Among others phenomena, AP-1 is involved in cell proliferation and proteolytic enzymes production. Regarding the differences of response to PKCδ overexpression of both models, it has been reported that this PKC isoform has a differential role, pro or anti tumorigenic, depending on cellular context. Citation Format: Maria I. Diaz Bessone, D. E. Berardi, S. M. Cirigliano, C. Flumian, E. D. Bal de Kier Joffe, L. B. Todaro, A. J. Urtreger. Differential response to retinoid treatment in mouse and human mammary tumor cell lines with alterations in protein kinase C (PKC) expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5203. doi:10.1158/1538-7445.AM2015-5203