Alejandro J. Urtreger

Protein kinase C δ enhances proliferation and survival of murine mammary cells

Protein kinase C (PKC) δ, a member of the novel family of PKC serine‐threonine kinases, has been implicated in negative regulation of proliferation and apoptosis in a large number of cell types, including breast cancer cell lines, and postulated as a tumor suppressor gene. In this study we show that in murine NMuMG mammary cells PKCδ promotes a mitogenic response. Overexpression of PKCδ in NMuMG cells leads to a significant increase in [3H]‐tymidine incorporation and cell proliferation, as well as enhanced extracellular signal‐regulated kinase (ERK)‐mitogen‐activated protein kinase (MAPK) activation. Activation of PKCδ with a phorbol ester leads to elevated cyclin D1 expression and an hyperphosphorylated Rb state. Surprisingly, ectopic expression of PKCδ conferred anchorage‐independent growth capacity to NMuMG cells. PKCδ overexpressors showed enhanced resistance to apoptotic stimuli, such as serum deprivation or doxorubicin treatment, an effect that correlates with hyperactivation of the Akt survival pathway. Our results provide evidence for a role of PKCδ as a positive modulator of proliferative and survival signals in immortalized mammary cells. The fact that PKCδ exerts differential responses depending on the cell context not only highlights the necessity to carefully understand the signaling events controlled by this PKC in each cell type but also suggests that we should be cautious in considering this kinase a target for cancer therapy.

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.

PKC delta (PKCδ) promotes tumoral progression of human ductal pancreatic cancer

Objective: Our objective was to study the role of protein kinase C delta (PKC&dgr;) 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 PKC&dgr; overexpression on in vivo and in vitro properties of human ductal carcinoma cell line PANC1. Results: Human ductal carcinomas showed PKC&dgr; overexpression compared with normal counterparts. In addition, in vitro PKC&dgr;-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 PKC&dgr;-PANC1. Interestingly, PKC&dgr;-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 PKC&dgr;-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 PKC&dgr;-PANC1 cells developed lung metastasis. Conclusion: Our results showed that the overexpression of PKC&dgr; 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.

Pharmacological strategies in lung cancer-induced cachexia: effects on muscle proteolysis, autophagy, structure, and weakness.

Cachexia is a relevant comorbid condition of chronic diseases including cancer. Inflammation, oxidative stress, autophagy, ubiquitin–proteasome system, nuclear factor (NF)‐κB, and mitogen‐activated protein kinases (MAPK) are involved in the pathophysiology of cancer cachexia. Currently available treatment is limited and data demonstrating effectiveness in in vivo models are lacking. Our objectives were to explore in respiratory and limb muscles of lung cancer (LC) cachectic mice whether proteasome, NF‐κB, and MAPK inhibitors improve muscle mass and function loss through several molecular mechanisms. Body and muscle weights, limb muscle force, protein degradation and the ubiquitin–proteasome system, signaling pathways, oxidative stress and inflammation, autophagy, contractile and functional proteins, myostatin and myogenin, and muscle structure were evaluated in the diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing cachectic mice (BALB/c), with and without concomitant treatment with NF‐κB (sulfasalazine), MAPK (U0126), and proteasome (bortezomib) inhibitors. Compared to control animals, in both respiratory and limb muscles of LC cachectic mice: muscle proteolysis, ubiquitinated proteins, autophagy, myostatin, protein oxidation, FoxO‐1, NF‐κB and MAPK signaling pathways, and muscle abnormalities were increased, while myosin, creatine kinase, myogenin, and slow‐ and fast‐twitch muscle fiber size were decreased. Pharmacological inhibition of NF‐κB and MAPK, but not the proteasome system, induced in cancer cachectic animals, a substantial restoration of muscle mass and force through a decrease in muscle protein oxidation and catabolism, myostatin, and autophagy, together with a greater content of myogenin, and contractile and functional proteins. Attenuation of MAPK and NF‐κB signaling pathway effects on muscles is beneficial in cancer‐induced cachexia. J. Cell. Physiol. 229: 1660–1672, 2014.

Characterization of F3II, a sarcomatoid mammary carcinoma cell line originated from a clonal subpopulation of a mouse adenocarcinoma.

We characterized a new mammary tumor cell line, F3II, previously established in vitro from a clonal subpopulation of the BALB/c transplantable mammary adenocarcinoma M3, moderately metastatic to lung. The F3II cell line has been passaged >50 times. It has grown as elongated cells adherent to the bottom of the flask. Cytogenetic studies showed that F3II cultures were nearly triploid. Tumor cells expressed fibronectin and showed high levels of cell‐surface urokinase, a key protease in invasion and metastasis. F3II cells grew as poorly differentiated, spindle‐cell carcinoma tumors (sarcomatoid carcinomas) with a prominent local invasiveness, a high angiogenic response, and a 90–100% incidence of lung metastases when inoculated s.c. into syngeneic mice. Ultrastructural and immunocytochemical analysis revealed characteristic features of carcinomas. Our data suggest that F3II is less differentiated and more aggressive than the original tumor line, supporting the notion that mammary carcinomas are heterogeneous neoplasms and contain subpopulations with diverse biologic behavior. The F3II mouse mammary sarcomatoid carcinoma line is a suitable model to examine antiinvasive, antiangiogenic, and antimetastatic agents.

Atypical protein kinase C‐ζ modulates clonogenicity, motility, and secretion of proteolytic enzymes in murine mammary cells

In this paper, we investigated whether protein kinase C‐ζ (PKCζ), a member of the atypical PKC family, induces phenotypic alterations associated with malignant transformation and tumor progression in mammary cells. The stable overexpression of PKCζ 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 PKCζ 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 PKCζ 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 PKCζ modulates several of the critical events involved in tumor development and dissemination through the activation of mitogen activated protein kinase (MAPK)/ERK pathway.

Downregulation of fibronectin transcription in highly metastatic adenocarcinoma cells

Silencing of fibronectin (FN) expression seems to be one of the key mechanisms underlying metastatic behaviour. An inverse correlation exists between FN expression levels and the metastatic potential of two related murine mammary adenocarcinomas, M3 and MM3. Primary cultures of M3 tumour, which is moderately metastatic to lung (40% incidence), show a conspicuous FN extracellular matrix (ECM) and high levels of FN mRNA, while primary cultures of the highly metastatic MM3 tumour (95% lung incidence) are negative for FN in immunofluorescence and show at least 40‐fold lower levels of FN mRNA, only detectable by RT‐PCR, with a different pattern of alternatively spliced EDI isoforms compared to M3 cells. We show that the FN promoter sequence is not altered in MM3 cells. Transfection experiments with CAT constructs indicate that silencing occurs at the transcriptional level, involving the 220‐bp proximal promoter region.

Mitochondrial dysfunction and therapeutic approaches in respiratory and limb muscles of cancer cachectic mice.

•  What is the central question of this study? We explored whether experimental cancer‐induced cachexia may alter mitochondrial respiratory chain (MRC) complexes and oxygen uptake in respiratory and peripheral muscles, and whether signalling pathways, proteasome and oxidative stress influence that process. •  What is the main finding and what is its importance? In cancer cachectic mice, MRC complexes and oxygen consumption were decreased in the diaphragm and gastrocnemius. Blockade of nuclear factor‐κB and mitogen‐activated protein kinase actions partly restored the muscle mass and force and corrected the MRC dysfunction, while concomitantly reducing tumour burden. Antioxidants improved mitochondrial oxygen consumption without eliciting effects on the loss of muscle mass and force or the tumour size, whereas bortezomib reduced tumour burden without influencing muscle mass and strength or MRC function.

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.

The neural cell adhesion molecule is involved in the metastatic capacity in a murine model of lung cancer.

Neural cell adhesion molecule (NCAM) is involved in cell growth, migration, and differentiation. Its expression and/or polysialylation appear to be deregulated in many different cancer types. We employed the lung tumor cell line LP07, syngeneic in BALB/c mice to investigate the role of NCAM in malignant progression. LP07 cells express the three main NCAM isoforms, all of them polysialylated. This cells line, pretreated with an anti‐NCAM antibody and inoculated intravenously (i.v.) into syngeneic mice, developed less and smaller lung metastases. In vitro studies showed that NCAM bound antibody inhibited cell growth, mainly due to an increase in apoptosis, associated with a decrease of cyclin D1 and enhanced expression of active caspase 3 and caspase 9. Anti‐NCAM‐treated LP07 cells showed impairment in their ability to migrate and adhere to several extracellular matrix components. Secreted uPA activity was also reduced. NCAM‐140 knocked‐down by siRNA in LP07 cells pretreated or not with anti‐NCAM showed an impaired metastasizing ability upon i.v. inoculation into mice. These results suggest that anti‐NCAM treatment could be mimicking homophilic trans‐interactions and NCAM‐140 knocked‐down impairs heterophilic interactions, both leading to inhibition of metastatic dissemination. The involvement of NCAM in lung tumor progression was confirmed in human NSCLC tumors. Sixty percent of the cases expressed NCAM at tumor cell level. A multivariate analysis indicated that NCAM expression was associated with a shorter overall survival in this homogeneous series of Stages I and II NSCLC patients. NCAM may be able to modulate mechanisms involved in lung carcinoma progression and represents an attractive target to control metastatic progression. Mol. Carcinog.