Cristina E. Rodríguez

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.

Breast cancer stem cells are involved in Trastuzumab resistance through the HER2 modulation in 3D culture.

Breast cancer human cells culture as spheroids develop autophagy and apoptosis, which promotes Trastuzumab resistance in HER2 overexpressing cells. Our aim was to study the association of the hostile environment developed in 3D with the breast cancer stem cells population and the HER2 modulation. Human mammary adenocarcinoma cell lines were cultured as spheroids using the hanging drop method. We generated hypoxia conditions by using a hypoxic chamber and CoCl2 treatment. Breast cancer stem cells were measured with mammosphere assays, the analysis of CD44 + CD24low population by flow cytometry and the pluripotent gene expression by RT‐qPCR. HER2 expression was evaluated by flow cytometry and Western blot. MTS assays were conducted to study cell viability. Hostil environment developed in spheroids, defined by hypoxia and autophagy, modulated the response to Trastuzumab. In HER2+ cells with acquired resistance, we observed an increase in the breast cancer stem cell population. In BT474 spheroids, Trastuzumab induced the acquisition of resistance, along with an increase in breast cancer stem cells. Also, in 3D culture conditions we determined a modulation in the HER2 expression. Moreover, breast cancer stem cells showed enhanced HER2 expression. Finally, cells without HER2 gene amplification cultured as spheroids were sensitive to Trastuzumab, diminishing HER2 expression and cancer stem cells. Our findings show that 3D architecture is able to modulate breast cancer stem cell population and HER2 distribution, modifying the cell response to Trastuzumab.

The Immune System As a New Possible Cell Target for AFP 464 in a Spontaneous Mammary Cancer Mouse Model

Aminoflavone (AFP 464, NSC 710464), an antitumor agent which recently entered phase II clinical trials, acts against estrogen‐positive breast cancer (ER+). AFP 464, which has a unique mechanism of action by activating aryl hydrocarbon receptor (AhR) signaling pathway, decreased tumor size, and growth rate in the estrogen dependent, Tamoxifen‐sensitive spontaneous M05 mouse model. Considering that AhR has recently emerged as a physiological regulator of the innate and adaptive immune responses, we investigated whether AFP 464 modulates the immune response in our mouse model. Studies on the effect of AFP 464 on the immune system were carried in BALB/c mice bearing M05 semi‐differentiated mammary adenocarcinomas that express estrogen and progesterone receptors. Splenic cells and tumor inflammatory infiltrates were studied by cytometric analyses. The modulation of splenocytes cytotoxic activity by AFP 464 was also evaluated. We further investigated the effects of AFP 464 on peritoneal macrophages by evaluating metalloproteinase, arginase, and iNOS activities. We found that AFP 464 increased splenic cytotoxic activity, diminished the number of systemic and local Treg lymphocytes, and MDSCs, and induced a M1 phenotype in peritoneal macrophages of M05 tumor bearing mice. Therefore, we conclude that AFP 464 modulates immune response which collaborates with its anti‐tumor activity. Our results place the immune system as a novel target for this anti‐cancer agent to strengthen the rationale for its inclusion in breast cancer treatment regimens. J. Cell. Biochem. 118: 2841–2849, 2017.

Abstract 4983: Aminoflavone acts as an immunomodulator of tumor growth in a breast cancer mouse model

Aminoflavone (AFP464, NSC 710464), an antitumor agent which recently entered phase II clinical trials, acts against estrogen-positive breast cancer (ER+). AFP464, which has a unique mechanism of action by activating aryl hydrocarbon receptor (AhR) signaling pathway, decreased tumor growth rate in an estrogen dependent, tamoxifen-sensitive mammary cancer murine model (M05) syngeneic in BALB/c developed in our animal facility. Considering that AhR has recently emerged as a physiological regulator of the innate and adaptive immune responses, it is our aim to investigate whether AFP464 modulates the immune response in our mouse model. Cytometric analyses of splenic cells obtained from AFP464-treated and control animals, at three different times after treatment (13, 21 and 41 days), revealed that MDSCs were lower in animals treated with AFP464 than in the vehicle control group. On the contrary (On the other hand) no differences in macrophages, NK, CD8+ T, and CD4+ T cells were observed between these two groups. We also found a diminished quantity of MDSCs in the inflammatory infiltrate, isolated from tumors treated with AFP464. According to these results, we also observed a higher cytotoxic activity of splenic cells from the AFP464-treated group compared with the control one (13 days: 1,50 ± 0,12 vs 0,74 ± 0,18; 21 days: 2,4 ± 0,3 vs 1,16 ± 0,2; 41 days: 2,97 ± 0,2. vs 1,07 ± 0,2 p Citation Format: Mariana Alejandra Callero, Cristina Elisa Rodriguez, Aldana Solimo, Elisa Bal de Kier Joffe, Andrea Loaiza Perez. Aminoflavone acts as an immunomodulator of tumor growth in a breast cancer mouse model. [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 4983.

Abstract 2881: Cytotoxic effect of trastuzumab on macrophage-infiltrated human mammary tumor spheroids

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The HER2 receptor, overexpressed in 20-25% of breast invasive tumors, is associated with low disease-free survival. Trastuzumab (Tz), monoclonal antibody anti HER2 is used as immunotherapy in HER2+ mammary tumors; however, 60% of patients are unresponsive to this therapy. The aim of our study is the analysis of the effect of Tz on the growth of human mammary tumor cells cultured in 3D (shperoids). The human adenocarcinoma cell line BT474 (HER2+) was used to generate spheroids grown in liquid RPMI1640 medium. One spheroid/well was seeded on a 1.5% agar layer, reaching 2.5 mm diameter at day 62 of culture. Differential expression of HER2, HIF-1, Bcl-2 and Ki67 were detected by immunohistochemistry. Proliferating, quiescent as well as apoptotic cells towards the hypoxic core of the tumor spheroids were identified. The effect of Tz (10 and 50 ug/ml/spheroid, 3 times/week) on the growth of 3D cultures of BT474 cells was evaluated, beginning with a volume of 0.23 ± 0.03 mm3 (day 0). Tz 10 ug/ml arrested spheroids growth since day 7 (3 doses) while Tz 50 ug/ml induced a significant reduction in the volume since the first dose (day 0) until day 27, when spheroids showed 71% of growth inhibition compared to untreated or human IgG controls (Tz 50= 0.12 ± 0.04 mm3 vs control= 0.45 ± 0.03 mm3, p<0.001). Spheroids showed lower sensitivity to the cytostatic effect of Tz 10 ug/ml when compared to the same cells cultured as monolayers. Tz exerted a direct and dose-dependent effect on BT474 tumor spheroids: while it was cytostatic at the lower dose (Tz10), it was cytotoxic at the higher (Tz50). Nitric oxide (NO) levels measured by Griess reaction in tumor spheroid supernatants were significantly higher upon Tz50 treatment compared to untreated controls (p<0.01), suggesting NO is involved in the direct cytotoxic mechanism of Tz. BT474 cells growing in monolayers responded to TZ with higher NO levels than 3D cultures. In order to assess whether immune system and tissue architecture have an impact on the Tz cytotoxic effect, we co-cultured single BT474 tumor spheroids with macrophages from human peripheral blood (ratio1:5). At 10 days co-culture, the macrophages had a type I response (M1) against tumor cells. In fact, we observed that the presence of macrophages in the culture increased the cytotoxicity of Tz. This period of time was not enough to switch macrophages to the M2 phenotype. Conditioned media from Tz50-treated BT474 spheroids decreased MMP9 production by macrophages cultures. We propose this 3D culture model as a useful tool to study susceptibility to trastuzumab therapy in HER2 + tumor cells, as well as to analyze the mechanisms of action and resistance of this treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2881. doi:1538-7445.AM2012-2881

Abstract 2930: The hostile microenvironment developed in 3D culture conditions induces Trastuzumab resistance involving cancer stem cells

HER2 is overexpressed in 20% invasive breast tumors and correlates with low free disease survival. Trastuzumab (Tz), monoclonal antibody anti HER2, is used to treat HER2+ tumors; however more than half of the patients are resistant or acquire resistance during treatment. Multicellular tumor spheroids are a 3D cell growth model that mimics the structure of in vivo avascular tumors, with heterogenic cell subpopulations developed due to differential oxygen and nutrient supply through the spheroid. We have previously demonstrated that HER2+ cells cultured as spheroids are more resistant to Tz than monolayers. We also observed that in spheroids Tz inhibited basal apoptosis and was capable of inducing autophagy, leading to Tz resistance. In addition, cancer stem cells (CSC), widely associated with chemotherapy resistance, were specifically targeted by Tz. The aim of this study was to analyze the resistance acquired in 3D and the impact of the CSC developed in these conditions. Since Tz-treated BT474 (HER2+) spheroids overexpressed the autophagy marker LC3, correlating with resistance to the treatment, we first evaluated these cells viability after autophagy inhibition with 3MA. We found that these resistant cells became susceptible to Tz after autophagy inhibition, decreasing viability by 33% (p To further study this 3D-induced resistance, we analyzed the CSCs subpopulation in spheroids, studying the CD44+CD24low cell phenotype by flow cytometry. We found that 15 days Tz treatment increased CD44+CD24low subpopulation by 1.5 fold compared to controls (p Then we analyzed HER2 expression in the spheroids and found 2 populations, HER2high and HER2low (37% vs 63%, respectively p Thus, we decided to study the 3D architecture in MCF7 cells, with no HER2 amplification and unresponsive to Tz inhibition. We analyzed HER2 by flow cytometry, observing HER2low expression in 82% of the cells that was reduced to 61% in Tz treated spheroids (p In conclusion, the hostile microenvironment in 3D has a key role in the development of resistance to Tz and could be overcome by autophagy inhibition. These conditions could favor an increase of CSCs unresponsive to Tz despite HER2 expression. Moreover, MCF7 results suggest that Tz is able to target CSCs developed in 3D. Citation Format: Cristina E. Rodriguez, Gabriel L. Fiszman, Elisa D. Bal de Kier joffe. The hostile microenvironment developed in 3D culture conditions induces Trastuzumab resistance involving cancer stem 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 2930.

Abstract P5-05-09: Autophagy in three dimensional cultures provides survival advantage against trastuzumab in HER2+ mammary adenocarcinoma cells

HER2 is overexpressed in 20-25% invasive breast tumors, and correlates with low free disease survival. Trastuzumab (Tz), monoclonal antibody anti HER2, is used to treat HER2+ tumors; however more than half of them are resistant or acquire resistance during treatment. Autophagy has been proposed as a tumoral escape mechanism. Multicellular tumor spheroids (MCS) are a model of cell growth in 3D that mimics the structure of in vivo avascular tumors. We have previously demonstrated that MCS present different subpopulations, with a gradient of proliferative, quiescent and apoptotic cells towards the center of the spheroid and these characteristics makes it more resistant to Tz than monolayers. The aim of this study was to analyze the role of autophagy in MCS growth and its relevance on the resistance of breast cancer cells to Tz. MCS of overexpressing HER2+ human mammary tumor cells (BT474 cell line) were cultured as cell suspensions on agar, one per well, and experiments were conducted when MCS reached 550 um initial size. When the autophagy marker LC3 was analyzed in MCS by Western blot, both LC3-I and LC3-II were significantly up-regulated compared with cells cultured as monolayers. The functional autophagic flux was confirmed by immunoblots of LC3 and p62 (SQSTM1/sequestosome1) in cells treated with Bafilomycin A1 (5 nM). MCS were fixed and included in paraffin to analyze the expression of LC3 and observed a differential localization of autophagic cells, increasing towards the center of the spheroid, correlating with the hypoxic population previously described. Uppon Tz adittion at a concentration of 50 ug/ml, a higher and uniform expression of LC3 was found in all the living cells. These observations were further supported by the finding that p62 was down-regulated in Tz treated spheroids in opposition to controls (commercial IgG). In 2D, Tz (1 ug/ml) also exerted LC3-II conversion and increase in autophagosomes formation. Autophagy inhibition by 3-methiladenine (3-MA) used at 1 mM, in combination with Tz decreased two fold vs Tz alone in monolayers (49% vs 76% cell viability respectively, p Citation Format: Cristina E Rodriguez, Sara Reidel, Elisa D Bal de Kier Joffe, Maria A Jasnis, Gabriel L Fiszman. Autophagy in three dimensional cultures provides survival advantage against trastuzumab in HER2+ mammary adenocarcinoma cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-09.

Abstract 349: Autophagy, cancer stem cells, and trastuzumab resistance in three dimensional cultures of HER2+ breast cancer cells

HER2 is overexpressed in 20% invasive breast tumors and correlates with low free disease survival. Trastuzumab (Tz), monoclonal antibody anti HER2, is used to treat HER2+ tumors; however more than half of the patients are resistant or acquire resistance during treatment. Multicellular tumor spheroids (MS) are a 3D growth model that mimics the structure of in vivo avascular tumors. Autophagy has been proposed as a tumoral escape mechanism. The cancer stem cell (CSC) hypothesis postulates that tumor growth is maintained by a small fraction of cells with unlimited proliferative potential that are also able to differentiate into cell populations that become the bulk of the tumor. CSCs are thought to be more resistant to chemotherapy and radiotherapy and related with tumor recurrence. We have previously demonstrated that MS present different subpopulations showing basal autophagy and apoptosis that increase towards the center of the spheroid, and these characteristics make them more resistant to Tz than monolayers. We also observed that Tz inhibited basal apoptosis in MS and was capable of inducing autophagy. Our aim was to analyze alternative mechanisms of resistance of HER2+ breast cancer cells against Tz. For this purpose, we used BT474 (HER2+) human breast cancer cell line, from which we developed a resistant subline (BT474-MR). MS were cultured one per well in agar covered plates. We observed that cells from BT474 MS treated with Tz for 15 days upregulated the autophagy marker LC3 and showed 100% Tz resistance when compared with cells from MS treated with non-related human IgG as control. BT474 cells were treated with the autophagy inhibitor 3-metyiladenine that was dose dependently cytotoxic both in 2D and 3D cultures. Interestingly, the higher concentration used was critical for spheroids survival but not for monolayers. BT474-MR cells were more sensitive to autophagy inhibition than parental cells (75.7 vs 100% cell viability, p Citation Format: Cristina E. Rodriguez, Sara Reidel, Maria Adela Jasnis, Elisa Bal de Kier joffe, Gabriel L. Fiszman. Autophagy, cancer stem cells, and trastuzumab resistance in three dimensional cultures of HER2+ breast cancer cells. [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 349. doi:10.1158/1538-7445.AM2015-349

Abstract 226: Trastuzumab effect on cell death and proliferation of HER2+ mammary adenocarcinoma cells using a tridimensional culture

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC HER2 is overexpressed in 20-25% invasive breast tumors, and associated with low free disease survival. Trastuzumab (Tz), monoclonal antibody anti HER2, is used as immunotherapy to treat HER2+ tumors; however more than half of them are resistant or acquire resistance during treatment. Cellular spheroids are a model of cell growth in 3D that mimic the structure of in vivo avascular tumors. We have previously demonstrated that tumor spheroids (TS) present different subpopulations, with a gradient of proliferative, quiescente and apoptotic cells from the outer rim towards the hypoxic core. The aim of this study was to analyze the biological characteristics of the different cell populations that constitute the TS and their response to Tz. TS of HER2+ human mammary tumor cells (cell line BT474) were cultured as cell suspensions on agar (hanging drop method) in 48-well plates (1 spheroid / well). Cell cycle at days 7, 14 and 21 of TS growth was analyzed by flow cytometry. A significant increase only in the G2/M phase (6.6%, 10.3% and 13.3% respectively) was detected. TS were incubated with Tz at a concentration of 50ug/ml; a commercial IgG (50ug/mL) was used as negative control. The effect on cell growth kinetics was evaluated with photographs taken at different times, observing a reduction of the spheroids volume until they reached a decrease by 71% at day 20 (p<0.01 vs controls). Tz induced an increase in the% of cells arrested in G1 (80.8% vs 64.7% control IgG, p<0.001) and a decrease in the proliferative subpopulation (7.4% vs 11.4%, p<0.05). A decrease in the% of apoptotic cells was also detected (Annexin V/IP). When we analyzed cell viability at different time points, we observed that Tz elicited changes in live to dead cells ratio. A reversion of the inhibitory effect of Tz on TS growth was observed after antibody withdrawal. Spheroids were fixed and included in paraffin to analyze the expression of HER2, pHER2, clived caspase 3, HIF-1a, p27, p21 and cyclinD1 by confocal microscopy. HER2 expression was not modulated by Tz; however, we observed lower expression of its activated receptor (pHER2). HIF-1a was expressed only in cells around the central core while clived caspase 3 was expressed in the central core. After Tz incubation, both markers were no longer detected. We can conclude that Tz exerted a potent antiproliferative and direct cytotoxic effect on BT474 tumor cells growing in 3D; these activities might be due to the partial inhibition of HER2 activation. Tz inhibited the formation of the hypoxic core and therefore the apoptotic subpopulation, generating thereby a smaller TS composed only of remaining Tz-resistant living cells. We propose that this model could be useful to study the mechanisms involved in the effect and on the resistance to Tz. Citation Format: Cristina E. Rodriguez, Sara Reidel, Luciana Moverer, Lina Marino, Elisa Bal de Kier Joffe, Maria A. Jasnis, Gabriel L. Fiszman. Trastuzumab effect on cell death and proliferation of HER2+ mammary adenocarcinoma cells using a tridimensional culture. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 226. doi:10.1158/1538-7445.AM2013-226