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

BACKGROUND: Lung 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. METHODS: The 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. RESULTS: We 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 ß-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. CONCLUSIONS: Our data provide new insights into CIGB-300 mechanism of action and suggest clinical potential on current NSCLC therapy.

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.

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

PURPOSE: Breast 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 METHODS: The 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Modulation of pancreatic tumor potential by overexpression of protein kinase C ß1.

OBJECTIVE: This study aimed to investigate whether the overexpression of protein kinase C ß1 (PKCß1) is able to modulate the malignant phenotype displayed by the human ductal pancreatic carcinoma cell line PANC1. METHODS: PKCß1 overexpression was achieved using a stable transfection approach. PANC1-PKCß1 and control cells were analyzed both in vitro and in vivo. RESULTS: PANC1-PKCß1 cells displayed a lower growth capacity associated with the down-regulation of the MEK/ERK pathway and cyclin expression. Furthermore, PKCß1 overexpression was associated with an enhancement of cell adhesion to fibronectin and with reduced migratory and invasive phenotypes. In agreement with these results, PANC1-PKCß1 cells showed an impaired ability to secrete proteolytic enzymes. We also found that PKCß1 overexpressing cells were more resistant to cell death induced by serum deprivation, an event associated with G0/G1 arrest and the modulation of PI3K/Akt and NF-κB pathways. Most notably, the overexpression of PKCß1 completely abolished the ability of PANC1 cells to induce tumors in nude mice. CONCLUSIONS: Our results established an important role for PKCß1 in PANC1 cells suggesting it would act as a suppressor of tumorigenic behavior in pancreatic cancer.

Contribution of individual PKC isoforms to breast cancer progression.

The protein kinase C (PKC) family of serine/threonine kinases has been intensively studied in cancer since their discovery as major receptors for the tumor-promoting phorbol esters. The contribution of each individual PKC isozyme to malignant transformation is only partially understood, but it is clear that each PKC plays different role in cancer progression. PKC deregulation is a common phenomenon observed in breast cancer, and PKC expression and localization are usually dynamically regulated during mammary gland differentiation and involution. In fact, the overexpression of several PKCs has been reported in malignant human breast tissue and breast cancer cell lines. In this review, we summarize the knowledge available on the specific roles of PKC isoforms in the development, progression, and metastatic dissemination of mammary cancer. We also discuss the role of PKC isoforms as therapeutic targets, and their potential as markers for prognosis or treatment response.

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.

PKC Delta (PKCdelta) promotes tumoral progression of human ductal pancreatic cancer.

OBJECTIVE: Our objective was to study the role of protein kinase C delta (PKCdelta) in the progression of human pancreatic carcinoma. METHODS: Protein kinase C delta expression in human ductal carcinoma (n = 22) was studied by immunohistochemistry. We analyzed the effect of PKCdelta overexpression on in vivo and in vitro properties of human ductal carcinoma cell line PANC1. RESULTS: Human ductal carcinomas showed PKCdelta overexpression compared with normal counterparts. In addition, in vitro PKCdelta-PANC1 cells showed increased anchorage-independent growth and higher resistance to serum starvation and to treatment with cytotoxic drugs. Using pharmacological inhibitors, we determined that phosphatidylinositol-3-kinase and extracellular receptor kinase pathways were involved in the proliferation of PKCdelta-PANC1. Interestingly, PKCdelta-PANC1 cells showed a less in vitro invasive ability and an impairment in their ability to migrate and to secrete the proteolytic enzyme matrix metalloproteinase-2. In vivo experiments indicated that PKCdelta-PANC1 cells were more tumorigenic, as they developed tumors with a significantly lower latency and a higher growth rate with respect to the tumors generated with control cells. Besides, only PKCdelta-PANC1 cells developed lung metastasis. CONCLUSION: Our results showed that the overexpression of PKCdelta in PANC1 cells induced a more malignant phenotype in vivo, probably through the modulation of cell proliferation and survival, involving phosphatidylinositol-3-kinase and extracellular receptor kinase signaling pathways.

Protein kinase C delta inhibits the production of proteolytic enzymes in murine mammary cells.

In previous studies we have determined that protein kinase C (PKC) delta, a widely expressed member of the novel PKC serine-threonine kinases, induces in vitro changes associated with the acquisition of a malignant phenotype in NMuMG murine mammary cells. In this study we show that PKCdelta overexpression significantly decreases urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) production, two proteases associated with migratory and invasive capacities. This effect is markedly enhanced by treatment with phorbol 12-myristate 13-acetate (PMA). On the other hand, depletion of PKCdelta using RNAi led to a marked increase in both uPA and MMP-9 secretion, suggesting a physiological role for PKCdelta in controlling protease secretion. The MEK-1 inhibitor PD98059 reverted the characteristic pattern of proteases secretion and phospho-ERK1/2 up-regulation observed in PKCdelta overexpressors, suggesting that the PKCdelta effect is mediated by the MEK/ERK pathway. Our results suggest a dual role for PKCdelta in murine mammary cell cancer progression. While this kinase clearly promotes mitogenesis and favors malignant transformation, it also down-modulates the secretion of proteases probably limiting metastatic dissemination.

Fibronectin is distinctly downregulated in murine mammary adenocarcinoma cells with high metastatic potential.

In the present work we used a murine mammary cancer model of two related adenocarcinomas with different lung metastasizing abilities, to compare their global gene expression profiles. Clontech Atlas mouse cDNA microarrays of primary cultured tumor cells were employed to identify genes that are modulated in the more metastatic variant MM3 relative to its parental tumor M3. A total of 88 from 1,176 genes were differentially expressed in MM3 primary cultures, most of them (n=86) were upregulated. Genes were grouped according to their functions as associated with signal transduction and transcription regulation (e.g. Stat1 and Zfp 92), with cell adhesion and motility (cadherin 1, fibronectin), with invasion and angiogenesis (uPA, 72 kDa MMP2), with the regulation of cell proliferation and cell death (cyclins G and A2, TNF), and also included growth factors and receptors, oncogenes and tumor suppressors genes (p107, TGFbeta2, TBR-I, PDGFR). Only 2 genes, TTF1 and fibronectin (FN), showed a significant downregulation. Notably FN expression, loss of which has been associated with a malignant phenotype, was reduced about 19-fold in the more metastatic MM3 cells. Previously known differences in expression patterns associated with the metastatic capacity of MM3 and M3 adenocarcinomas, including downregulation of FN or upregulated expression of TGFbeta and proteases, were confirmed by the array data. The fact that FN was one of the only two genes significantly down-regulated out of the 1,176 genes analyzed stresses the hypothesis that FN may behave as an important metastasis suppressor gene in mammary cancer.

Atypical protein kinase C-zeta modulates clonogenicity, motility, and secretion of proteolytic enzymes in murine mammary cells.

In this paper, we investigated whether protein kinase C-zeta (PKC zeta), a member of the atypical PKC family, induces phenotypic alterations associated with malignant transformation and tumor progression in mammary cells. The stable overexpression of PKC zeta in immortalized mammary epithelial cells (NMuMG), activates the mitogenic extracellular signal-regulated kinase (ERK) pathway, enhanced clonal cell growth and exerts profound effects on proteases secretion. The effect on proteases expression seems to be specific for urokinase-type plasminogen activator and metalloproteinase-9 (MMP-9) because no modulation in MMP-2 and MMP-3 production could be detected. In addition, our experiments demonstrated that PKC zeta overexpression markedly altered the adhesive, spreading, and migratory abilities of NMuMG cells. The overexpression of this enzyme was not sufficient to confer an anchorage-independent growth capacity. An extensive mutational analysis of PKC zeta revealed that the effects observed in NMuMG cells were strictly dependent on the kinase (catalytic) domain of the enzyme. Taken together, these results suggest that in mammary cells PKC zeta modulates several of the critical events involved in tumor development and dissemination through the activation of mitogen activated protein kinase (MAPK)/ERK pathway.