Elisa Bal de Kier Joffé

PLASMA MMP-9 (92 kDa-MMP) ACTIVITY IS USEFUL IN THE FOLLOW-UP AND IN THE ASSESSMENT OF PROGNOSIS IN BREAST CANCER PATIENTS

Previously we determined that plasma MMP‐9 activity was significantly elevated in breast cancer patients compared to benign mammary pathologies and healthy controls. Now we analyzed its potential usefulness in the follow‐up and in the prognosis of these patients. MMP‐9 activity was measured by gelatin quantitative zymography in the euglobulin plasma fraction of 46 breast cancer patients in a 38‐month follow‐up study. Blood samples were obtained before surgery (S1), 1 month after (S2) and every 3 months. The relapse‐free survival (RFS) and overall survival (OS) analysis was performed along 56 months in 113 patients using the Kaplan‐Meier curves and Cox analysis. In 63% of the S2 analyzed, MMP‐9 decreased after surgery. In 44 patients evaluated during the adjuvant period who developed a complete response, MMP‐9 decreased compared to their S1, whereas 2 patients showed an enhancement in correlation with lack of response. Further analysis indicated that in all patients who never showed evidence of recurrence, plasma MMP‐9 activity remained low, but it increased 1 to 8 months preceding the clinical detection of progression in those patients who relapsed. Kaplan‐Meier curves indicated that high levels of plasma MMP‐9 activity at the moment of breast cancer diagnosis were associated with a worse OS rate. Cox analysis showed it was not associated with tumor stage or patients age. Our results, which show a good correlation between plasma MMP‐9 activity and the clinical status of each patient, suggest its usefulness as a marker both in the follow‐up and in the prognosis of breast cancer patients.

The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through β1 integrin

Tamoxifen resistance has been largely attributed to genetic alterations in the epithelial tumor cells themselves, such as overexpression of HER-2/Neu. However, in the clinic, only about 15–20% of cases of HER-2/Neu amplification has actually been correlated to the acquisition of endocrine resistance, suggesting that other mechanisms must be involved as well. Using the epithelial LM05-E and the fibroblastic LM05-F cell lines, derived from the estrogen dependent spontaneous M05 mouse mammary tumor, as well as MCF-7 cells, we analyzed whether soluble stromal factors or extracellular matrix components protected against tamoxifen induced cell death. Involvement of signaling pathways was determined by using specific inhibitors and western blot, and phosphorylation of the estrogen receptor alpha by western blot and immunofluorescence. Soluble factors produced by the fibroblastic cells protect the epithelial tumor cells from tamoxifen-induced cell death through a mechanism that involves EGFR and matrix metalloproteinases upstream of PI3K/AKT. Exogenous fibronectin by itself confers endocrine resistance through interaction with β1 integrin and activation of PI3K/AKT and MAPK/ERK 1/2 pathways. The conferred resistance is reversed by blocking β1 integrin. We show also that treatment with both conditioned medium and fibronectin leads to the phosphorylation of the estrogen receptor at serine-118, suggesting stromal factors as modulators of ER activity. Our results show that the tumor microenvironment can modulate tamoxifen resistance, providing an alternative explanation for why patients become refractory to hormone-therapy.

RalA requirement for v-Src- and v-Ras-induced tumorigenicity and overproduction of urokinase-type plasminogen activator: involvement of metalloproteases.

Overproduction of urokinase-type plasminogen activator (uPA) and metalloproteases (MMPs) is strongly correlated with tumorigenicity and with invasive and metastatic phenotypes of human and experimental tumors. We demonstrated previously that overproduction of uPA in tumor cells is mediated by a phospholipase D (PLD)- and protein kinase C-dependent mechanism. The oncogenic stimulus of v-Src and v-Ras results in the activation of PLD, which is dependent upon the monomeric GTPase RalA. We have therefore investigated whether RalA plays a role in uPA and MMP overproduction that is observed in response to oncogenic signals. We report here that NIH3T3 cells transformed by both v-Src and v-Ras, constitutively overproduce uPA and that expression of a dominant negative RalA mutant (S28N) blocks overproduction of uPA in both the v-Src-and v-Ras-transformed cells. v-Src and v-Ras also induced an upregulation of the activity of MMP-2 and MMP-9 as detected by zymograms, however only the v-Src induction correlated with MMP protein levels detected by Western blot analysis. The dominant negative RalA mutant blocked increased MMP-2 and 9 overproduction induced by v-Src, but not the increased activity of MMP-2 and 9 induced by v-Ras. And, consistent with a role for the RalA/PLD pathway in mitogenesis and tumor development, the dominant negative RalA mutant completely blocked tumor formation by v-Src- and v-Ras-transformed NIH3T3 cells injected subcutaneously in syngeneic mice. The data presented here implicate RalA and PLD as signaling mediators for tumor formation and protease production by transformed cells.

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.

Plasma metalloproteinase activity is enhanced in the euglobulin fraction of breast and lung cancer patients

Matrix metalloproteinases (MMP) have been implicated in tumor invasion and metastasis. We verified, by gelatin zymography, MMP activity in the euglobulin plasma fraction of 82 healthy controls, 66 patients with benign diseases and 149 patients with breast, lung, colon or brain cancer. The euglobulin fractions assayed showed 4 gelatinolytic bands of 62, 92, 120 and 200 kDa. The median (Md) value for 92 kDa‐MMP activity was significantly increased in breast (Md 1.34 arbitrary units [AU]/ml plasma, range 0.0–7.2) and lung cancer patients (Md 1.43 AU/ml, range 0.0–3.6) compared with the controls (Md 0.48 AU/ml, range 0.0–1.8). Patients with colon cancer or gliomas presented values of MMP‐9 similar to those of the healthy population. Multivariate analysis indicated that plasma MMP‐9 activity was not predicted by the known clinicopathological parameters such as age, stage, tumor size, number of positive lymph nodes, histologic grade, histologic type, nuclear grade or mitotic index. Lung cancer patients also presented high values of MMP‐9 (Md 1.43, range 0.0–3.6 [n = 26]), without association with tumor stage or histologic type. The levels of 92 kDa‐MMP activity in the plasma euglobulin fraction could be a potentially useful tumor marker in breast and lung cancer. Int. J. Cancer 89:389–394, 2000.

Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria

Mitochondria are major cellular sources of hydrogen peroxide (H2O2), the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H2O2 concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H2O2 due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs) orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H2O2 yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 µM H2O2 increased cell proliferation in ERK1/2-dependent manner whereas high 50 µM H2O2 arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei), the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs) facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H2O2 level. Like this, high H2O2 or directed mutation of redox-sensitive ERK2 Cys214 impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to increase H2O2 yield should disrupt synchronized MAPK oxidations and the regulation of cell cycle leading cells to remain in a proliferating phenotype.

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.

Effects of synthetic urokinase inhibitors on local invasion and metastasis in a murine mammary tumor model.

Urokinase-type plasminogen activator (uPA) initiates an extracellular proteolytic cascade with which invasive cells eliminate barriers to movement. We have evaluated the antiinvasive and antimetastatic properties of two recently developed synthetic uPA inhibitors, B428 and B623, in a BALB/c mouse mammary carcinoma model. We used the F3II and M3 tumor cell lines, previously described by our laboratory.In vitro, noncytotoxic concentrations of B428 or B623 inhibited secreted and cell-associated uPA activity produced by tumor cells and blocked uPA-mediated whole tumor cell degradation of fibronectin, allowing deposition of extracellular fibronectin fibrils.In vivo, administration of compounds was not associated with overt toxic effects. Daily i.p. treatment with B428 (20 mg/kg/day) or B623 (7.5 mg/kg/day) for 2 weeks, beginning after tumor take, markedly blocked the invasion of the muscle and adipose layers of the subcutis and dermis in mice bearing highly invasive F3II tumors. However, these compounds neither inhibited tumor-induced angiogenesis nor reduced the incidence of spontaneous lung metastasis. Moreover, B623 enhanced the formation of experimental lung metastasis. Our results suggest that synthetic uPA inhibitors act as potent antiinvasiveness agentsin vivo but may be unable to control progression of the metastatic disease in the present mammary tumor model.

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.