Brefeldin A triggers apoptosis associated with mitochondrial breach and enhances HA14-1- and anti-Fas-mediated cell killing in follicular lymphoma cells

Follicular lymphoma (FL) remains a fatal disease of increasing worldwide incidence. Since patients with FL eventually develop resistance to conventional anticancer agents, and due to BCL-2 overexpression present with profoundly compromised execution of mitochondrial pathway of apoptosis, targeting alternative pathways of cell demise may appear therapeutically beneficial. Herein we report for the first time the effects of an ER-Golgi transport inhibitor, Brefeldin A (BFA), alone and in combination with a small molecule Bcl-2 inhibitor HA14-1 or agonistic anti-Fas mAb, in the recently established human FL cell lines. All cell lines tested were sensitive to BFA-induced cytotoxicity and apoptosis. Moreover BFA-induced cell death was associated with profound ER stress, mitochondrial breach and subsequent caspase cascade activation, including caspase 2 activation. Interestingly, BFA-induced ER stress did not result in appearance of autophagic morphology in FL cells. Of importance, small molecule Bcl-2 antagonist, HA14-1 and agonistic anti-Fas mAb significantly enhanced BFA-mediated cytotoxicity and apoptosis, revealing novel and previously unexplored means to enhance ER stress-mediated cell killing in follicular lymphoma cells.     

Inhibition of the antiproliferative effect of TGFbeta by EGF in primary human ovarian cancer cells

The majority of ovarian cancers (OCs) arise from the ovarian surface epithelium (OSE). Proliferation of the OSE can be regulated by a number of autocrine and paracrine factors, including transforming growth factor beta (TGFbeta). Defects in the TGFbeta signaling pathway have been implicated in a number of cancers, including ovarian. We previously found that the TGFbeta signaling pathway is intact and functional in primary human OC cells, and that these cells stop growing in response to TGFbeta. Ovarian cancer cells in vivo are exposed to TGFbeta, yet continue to proliferate, therefore, mechanisms must exist to inhibit TGFbeta signaling contributing to uncontrolled cellular proliferation. Numerous signaling pathways converge with the TGFbeta pathway to modulate its effects, including signaling induced by epidermal growth factor (EGF). We hypothesized that EGF can modulate TGFbeta signaling and contribute to uncontrolled cellular proliferation of OC cells. Our results show that EGF abrogates the antiproliferative effect of TGFbeta. EGF does not modulate TGFbeta signaling by inhibiting receptor-activated Smad (R-Smad) phosphorylation or nuclear translocation. Rather, EGF decreases TGFbeta-induced mRNA expression of the cell cycle regulator, p15(INK4B), contributing to decreased sensitivity of OC cells to the antiproliferative effect of TGFbeta.     

Induction of p16ink4a and p19ARF by TGFbeta1 contributes to growth arrest and senescence response in mouse keratinocytes

TGFbeta1 acts as a potent negative regulator of the cell cycle and tumor suppressor in part through induction of cyclin dependent kinase inhibitors p15(ink4b), p21, and p57. We previously showed that primary mouse epidermal keratinocytes (MEK) expressing a v-ras(Ha) oncogene undergo hyperproliferation followed by growth arrest and senescence that was dependent on TGFbeta1 signaling and associated with increased levels of p16(ink4a) and p19(ARF). Here we show that the induction of both p16(ink4a) and p19(ARF) in v-ras(Ha) expressing keratinocytes is dependent on TGFbeta1 signaling, as TGFbeta1 treatment or Smad3 overexpression induces both p16(ink4a) and p19(ARF) protein and mRNA, while Smad3 depletion or Smad7 overexpression blocks induction. Genetic ablation of the cdkn2a (ink4a/arf) locus reduced sensitivity to TGFbeta1 mediated cell cycle arrest and induction of senescence suggesting that alteration of TGFbeta1 responses may be an additional pathway impacted by the inactivation of cdkn2a locus during tumor development.     

Hair follicle defects and squamous cell carcinoma formation in Smad4 conditional knockout mouse skin

Smad4 is the common mediator for TGFbeta signals, which play important functions in many biological processes. To study the role of Smad4 in skin development and epidermal tumorigenesis, we disrupted this gene in skin using the Cre-loxP approach. We showed that absence of Smad4 blocked hair follicle differentiation and cycling, leading to a progressive hair loss of mutant (MT) mice. MT hair follicles exhibited diminished expression of Lef1, and increased proliferative cells in the outer root sheath. Additionally, the skin of MT mice exhibited increased proliferation of basal keratinocytes and epidermal hyperplasia. Furthermore, we provide evidence that the absence of Smad4 resulted in a block of both TGFbeta and bone morphogenetic protein (BMP) signaling pathways, including p21, a well-known cyclin-dependent kinase inhibitor. Consequently, all MT mice developed spontaneous malignant skin tumors from 3 months to 13 months of age. The majority of tumors are malignant squamous cell carcinomas. A most notable finding is that tumorigenesis is accompanied by inactivation of phosphatase and tensin homolog deleted on chromosome 10 (Pten), activation of AKT, fast proliferation and nuclear accumulation of cyclin D1. These observations revealed the essential functions of Smad4-mediated signals in repressing skin tumor formation through the TGFbeta/BMP pathway, which interacts with the Pten signaling pathway.     

TGFbeta1 represses proliferation of pancreatic carcinoma cells which correlates with Smad4-independent inhibition of ERK activation

Transforming growth factor beta (TGFbeta) is a tumor suppressor acting as inhibitor of cell cycle progression of epithelial cells. We show that treatment of the pancreatic carcinoma cell lines PANC-1 and BxPC-3 with TGFbeta1 inhibits both growth factor-induced activation of the extracellular signal-regulated kinase 2 (ERK2) and translocation of the kinase to the nucleus. TGFbeta1 causes a concentration-dependent reduction of cell proliferation in both cell lines. By measuring ERK activation, we can show that TGFbeta1 is able to repress ERK activation induced by mitogenic stimuli such as EGF. This inhibitory effect of TGFbeta1 is not mediated by suppression of Ras or c-Raf-1 activation, but mediated by TGFbeta1-induced activation of a serine-threonine phosphatase, as demonstrated by inhibition of phosphatases by treatment with okadaic acid. Results obtained in the Smad4-deficient pancreatic carcinoma cell line BxPC-3, demonstrate that TGFbeta1-induced growth inhibition is mediated by a Smad4-independent prevention of ERK2 activation. In contrast to the effects of TGFbeta1 on epithelial cells, mesenchymal NIH3T3 fibroblasts exhibit elevated ERK2 activation and increased cell proliferation in response to TGFbeta1 treatment. Smad4-independent phosphatase-mediated inhibition of mitogen-activated ERK2 represents a novel effector pathway contributing to suppression of epithelial pancreatic carcinoma cell proliferation by TGFbeta1, in addition to the well-known Smad-induced tumor suppressor activity of TGFbeta. Oncogene (2000) 19, 4531 - 4541.     

PI3K signaling pathway in normal B cells and indolent B-cell malignancies

In chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphomas (NHLs), B-cell receptor signaling leads to activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Idelalisib, a PI3Kδ inhibitor was approved in 2014 by the US Food and Drug Administration (FDA) in combination with rituximab for the treatment of patients with CLL for whom single-agent rituximab would be considered appropriate and as a single agent for patients with relapsed small lymphocytic lymphoma (SLL) and relapsed follicular lymphoma (FL). Following its approval, several trials investigating various PI3Kδ inhibitors as single agents or in combination with chemoimmunotherapy or other molecular targeted agents in CLL and indolent NHL (iNHL) have uncovered some severe autoimmune related toxicities. This review discusses and summarizes the biologic basis and the clinical experience of the PI3Kδ inhibitors in indolent B-cell malignancies.     

The type III TGF-beta receptor signals through both Smad3 and the p38 MAP kinase pathways to contribute to inhibition of cell proliferation

Transforming growth factor beta (TGFbeta) has an important role as a negative regulator of cellular proliferation. The type III transforming growth factor beta receptor (TbetaRIII) has an emerging role as both a TGFbeta superfamily co-receptor and in mediating signaling through its cytoplasmic domain. In L6 myoblasts, TbetaRIII expression enhanced TGFbeta1-mediated growth inhibition, with this effect mediated, in part, by the TbetaRIII cytoplasmic domain. The effects of TbetaRIII were not due to altered ligand presentation or to differences in Smad2 phosphorylation. Instead, TbetaRIII specifically increased Smad3 phosphorylation, both basal and TGFbeta-stimulated Smad3 nuclear localization and Smad3-dependent activation of reporter genes independent of its cytoplasmic domain. Conversely, SB431542, a type I transforming growth factor beta receptor (TbetaRI) inhibitor, as well as dominant-negative Smad3 specifically and significantly abrogated the effects of TbetaRIII on TGFbeta1-mediated inhibition of proliferation. TbetaRIII also specifically increased p38 phosphorylation, and SB203580, a p38 kinase inhibitor, specifically and significantly abrogated the effects of TbetaRIII/TGFbeta1-mediated inhibition of proliferation in L6 myoblasts and in primary human epithelial cells. Importantly, treatment with the TbetaRI and p38 inhibitors together had additive effects on abrogating TbetaRIII/TGFbeta1-mediated inhibition of proliferation. In a reciprocal manner, short hairpin RNA-mediated knockdown of endogenous TbetaRIII in various human epithelial cells attenuated TGFbeta1-mediated inhibition of proliferation. Taken together, these data demonstrate that TbetaRIII contributes to and enhances TGFbeta-mediated growth inhibition through both TbetaRI/Smad3-dependent and p38 mitogen-activated protein kinase pathways.     

Smad2 mediates Erk1/2 activation by TGF-beta1 in suspended, but not in adherent, gastric carcinoma cells

Integrin-mediated cell adhesion enables cells to respond to extracellular stimuli for diverse cellular functions including proliferation, leading to differential biological activities from cells in suspension. Integrins can transduce signals (directly) to intracellular molecules and also collaborate with other membrane receptor-mediated signal pathways, including TGF-beta1 pathway. TGF-beta1 induces growth inhibition in epithelial cells and is known to transduce intracellular signaling in Smad-dependent or -independent manner. Currently effects of cell adhesion status on the TGF-beta1-mediated Erk1/2 regulation and on its Smad-(in)dependency are not known. In this study, we examined effects of cell adhesion status on the TGF-beta1-mediated Erk1/2 regulation, and roles of Smad proteins on the cell adhesion-mediated effects, using a gastric carcinoma cell variant. First, we found that cell adhesion-dependent Erk1/2 activation responded differentially to TGF-beta1, depending on cell adhesion status; TGF-beta1 treatment resulted in activation of Erk1/2 in suspended cells, whereas a decrease was noted in adherent cells. This activation of Erk1/2 by TGF-beta1 in suspension was more enhanced by an overexpression of Smad2, but not of other Smads 2, 4, and 7, but abolished by a Smad2 reduction via an introduction of its siRNA. In contrast, PKB/Akt regulation by TGF-beta1 was not different in suspension or in adhesion, and Smad7, but not the other Smads, activated PKB/Akt phosphorylation on TGF-beta1 treatment, indicating a specificity of Smad2-mediated and cell adhesion status-dependent activation of Erk1/2 activity.     

Activation of bone morphogenetic protein signaling by a Gemini vitamin D3 analogue is mediated by Ras/protein kinase C alpha

Bone morphogenetic proteins (BMP) are members of the transforming growth factor-beta superfamily, and they play an important role for embryonic development, for bone and cartilage formation, and during carcinogenesis. We have previously shown that the novel Gemini vitamin D(3) analogue, Ro-438-3582 [Ro3582; 1 alpha,25-dihydroxy-20S,21(3-hydroxy-3-methylbutyl)-23-yne-26,27-hexafluorocholecalciferol], inhibited cell proliferation and activated the BMP/Smad signaling pathway in MCF10AT1 breast epithelial cells. In this report, we investigated the upstream signaling pathways responsible for the activation of BMP/Smad signaling by Ro3582. Among seven different serine/threonine kinase inhibitors that we tested, protein kinase C (PKC) inhibitors blocked the effects of Ro3582 on the phosphorylation of Smad1/5, mRNA synthesis for BMP-2 and BMP-6, and cell growth in MCF10AT1 cells. Overexpression of PKC alpha, but not PKC epsilon, PKC delta or PKC zeta isoforms, increased Ro3582-induced phosphorylation of Smad1/5, suggesting that PKC alpha mediates the activation of Smad signaling and inhibition of cell proliferation. Interestingly, the activation of Smad signaling by Ro3582 was shown in Ha-ras-transfected MCF10AT1 cells, but not in the parent cell line (MCF10A without Ras). Inhibiting Ras activity blocked the translocation of PKC alpha to the plasma membrane and the phosphorylation of Smad1/5 induced by Ro3582, indicating that Ras is necessary for the activation of PKC alpha and Smad signaling. In conclusion, Ro3582 inhibits cell proliferation and activates BMP/Smad signaling via a Ras and PKC alpha pathway in breast epithelial cells.     

Cancer-associated transforming growth factor beta type II receptor gene mutant causes activation of bone morphogenic protein-Smads and invasive phenotype

Transforming growth factor beta (TGFbeta) plays a key role in maintaining tissue homeostasis by inducing cell cycle arrest, differentiation and apoptosis, and ensuring genomic integrity. Furthermore, TGFbeta orchestrates the response to tissue injury and mediates repair by inducing epithelial to mesenchymal transition and by stimulating cell motility and invasiveness. Although loss of the homeostatic activity of TGFbeta occurs early on in tumor development, many advanced cancers have coopted the tissue repair function to enhance their metastatic phenotype. How these two functions of TGFbeta become uncoupled during cancer development remains poorly understood. Here, we show that, in human keratinocytes, TGFbeta induces phosphorylation of Smad2 and Smad3 as well as Smad1 and Smad5 and that both pathways are dependent on the kinase activities of the type I and II TGFbeta receptors (T beta R). Moreover, cancer-associated missense mutations of the T beta RII gene (TGFBR2) are associated with at least two different phenotypes. One type of mutant (TGFBR2(E526Q)) is associated with loss of kinase activity and all signaling functions. In contrast, a second mutant (TGFBR2(R537P)) is associated with high intrinsic kinase activity, loss of Smad2/3 activation, and constitutive activation of Smad1/5. Furthermore, this TGFBR2 mutant endows the carcinoma cells with a highly motile and invasive fibroblastoid phenotype. This activated phenotype is T beta RI (Alk-5) independent and can be reversed by the action of a dual T beta RI and T beta RII kinase inhibitor. Thus, identification of such activated T beta RII receptor mutations in tumors may have direct implications for appropriately targeting these cancers with selective therapeutic agents.     

Tumor suppressor and oncogene actions of TGFbeta1 occur early in skin carcinogenesis and are mediated by Smad3

Interactions between TGFbeta1 and ras signaling pathways play an important role in cancer development. Here we show that in primary mouse keratinocytes, v-ras(Ha) does not block the early biochemical events of TGFbeta1 signal transduction but does alter global TGFbeta1 mediated gene expression in a gene specific manner. Expression of Smad3 dependent TGFbeta1 early response genes and the TGFbeta1 cytostatic gene expression response were not altered by v-ras(Ha) consistent with an intact TGFbeta1 growth arrest. However, TGFbeta1 and v-ras(Ha) cause significant alteration in genes regulating matrix remodeling as the TGFbeta1 induction of extracellular matrix genes was blocked by v-ras(Ha) but specific matrix proteases associated with cancer progression were elevated. Smad3 deletion in keratinocytes repressed normal differentiation maker expression and caused expression of Keratin 8 a simple epithelial keratin and marker of malignant conversion. Smad3 was required for the TGFbeta1 cytostatic response in v-ras(Ha) keratinocytes, but also for protease induction, keratinocyte attachment and migration. These results show that pro-oncogenic activities of TGFbeta1 can occur early in carcinogenesis before loss of its tumor suppressive function and that selective regulation rather than complete inactivation of Smad3 function may be crucial for tumor progression.     

Novel murine B-cell lymphoma/leukemia model to study BCL2-driven oncogenesis

The BCL-2 family has been implicated in the pathogenesis of various hematopoietic malignancies, including follicular non-Hodgkin lymphoma and B-cell chronic lymphocytic leukemia. To identify genes that act synergistically in BCL2-enforced leukemogenesis, we developed a murine B-cell lymphoma/leukemia model based on the IL-3-dependent Balb/C pro-B line (FL5.12). FL5.12 cells were stably transfected with antiapoptotic BCL-2 alone or in combination with proapoptotic BAX or nonfunctional mutant BAX, thereby creating various levels of imbalance within the BCL-2 family. Transfectants were intravenously injected into normal Balb/C mice. Whereas FL5.12 cells did not provoke leukemia, mice injected with stable transfectants died of leukemia over time. Disease incidence and latency time depended on the degree of imbalance in the BCL-2 family, supporting a model whereby BCL2 drives tumorigenesis. All mice presented with hepatosplenomegaly and leukemic FL5.12 cells in peripheral blood and bone marrow compartments. Leukemic conversion was accompanied by secondary genetic aberrations leading to clonal IL-3-responsive leukemia. Cellular transformation was independent of alterations in c-Myc or downstream apoptotic pathway. Leukemic clones retained a normal DNA damage response leading to elevated P53 and P21 levels and cell cycle arrest upon irradiation. In conclusion, our mouse model may prove a valuable tool to identify genes that cooperate in BCL2-enforced lymphoma/leukemogenesis.