Mustapha Kandouz

Gap junctions and connexins as therapeutic targets in cancer

Importance of the field: Connexins (Cxs) and gap junctional intercellular communications (GJICs) play roles in cancer development, growth and metastasis. Experimental studies suggest that targeting Cxs may be a novel technique, either to inhibit tumor cell growth directly or to sensitize to various therapies. Areas covered in this review: A brief introduction to the role of Cxs in cancer. The focus is mainly on data available in the literature regarding therapeutic aspects. What the reader will gain: This article reviews the various strategies that take advantage of gap junctions and connexins to eliminate cancer cells, including use of the bystander effect (BE) in gene therapy, the effect of connexins on chemosensitization, the role of apoptotic processes and interactions with the microenvironment. Attempts to restore connexin expression at the transcriptional and post-transcriptional levels are described, as well as promising strategies recently explored. The potential and limitations of the approaches are discussed. Take home message: Connexins have multiple facets, singly, in hemichannel complexes, in gap junctions or interacting with different proteins. The regulation of their expression is not fully resolved and selective manipulation of Cxs expression is therefore a challenge. Although the therapeutic potential of connexins is undeniable, more effort is needed to study the regulation and functions of these proteins.

Antagonism between estradiol and progestin on Bcl‐2 expression in breast‐cancer cells

Bcl‐2 is a key protein involved in the control of apoptosis. Our previous studies on breast and endometrium indicated hormonal regulation of bcl‐2 in these tissues. In the present work we have analyzed Bcl‐2 and Bax protein expressions in MCF‐7 and T47‐D, 2 hormone‐dependent breast‐cancer cell lines, by immunoblots. Estradiol markedly increased Bcl‐2 protein content, both in short‐ and in long‐term treatments of MCF‐7 cells. Two types of anti‐estrogens (4‐hydroxytamoxifen and RU 58668) were able to reverse this effect. Also, a synthetic progestin (ORG 2058) was able to decrease the Bcl‐2 level in T47‐D cells. The level of Bax protein, however, was not affected in the same conditions of hormonal treatments. The level of Bcl‐2 expression was 4.5‐fold higher in MCF‐7 than in MDA‐MB 231 (an estradiol‐independent cell line). From these results, we infer the existence of hormonal regulation of Bcl‐2 expression and evoke a novel role for estradiol and progestin in the genesis of breast cancer.

Connexin43 pseudogene is expressed in tumor cells and inhibits growth.

Pseudogenes are classically thought of as nonfunctional DNA sequences due to their inability to be translated, or to produce a functional protein. Gap junctions, a multiprotein complex made of proteins called connexins, are involved in intercellular communication and are deregulated in many cancers. Connexin43 (Cx43) is the only connexin for which a pseudogene has been reported so far. The Cx43 pseudogene (ΨCx43) has all of the features of an expressed gene. We identified the presence of a ΨCx43 mRNA transcript in several cancer cell lines and in none of the normal mammary epithelial cells studied. Using an in vitro translation assay, we found that the ΨCx43 coding plasmid could be translated into a 43 kDa protein. This was further confirmed by expressing a ΨCx43-green fluorescence protein fusion protein in breast cancer MCF-7 cells. We then examined the functional significance of the ΨCx43. In both MTT growth and colony formation assays, significant growth inhibition was observed, a feature common to cells overexpressing the Cx43 gene. However, using a scrape-loading assay, we could not detect any effect on gap junctional intercellular communication. Based on our findings, ΨCx43 joins and enlarges the thus far restricted group of functionally transcribed and translated pseudogenes.

E6/E7 of HPV Type 16 Promotes Cell Invasion and Metastasis of Human Breast Cancer Cells

Human papillomaviruses (HPVs) could be important risk factors for breast carcinogenesis and metastasis, as roughly 50% of breast cancers are positive for high-risk HPVs. To determine the role of high-risk HPVs in human breast carcinogenesis and metastasis, we examined the effect of E6/E7 of HPV type 16 in two non-invasive breast cancer cell lines, MCF7 and BT20. We report that E6/E7 of HPV type 16 induces cell invasive and metastatic abilities of MCF7 and BT20 in vitro and in vivo, respectively, in comparison with the wild type cells. This is accompanied by an up-regulation of Id-1, a family member of helix-loop-helix (HLH) transcription factors, in MCF7 and BT20 cell lines which express E6/E7. Earlier studies have reported that Id-1 regulates cell invasion and metastasis of human breast cancer cells. To gauge the role of Id-1 in cell invasion and metastasis induced by E6/E7 of HPV type 16, we investigated the effect of E6/E7 in mouse normal embryonic fibroblast (NEF) and knockout Id-1 (Id-1-/-) cells. We establish that E6/E7 induces cell invasive ability in NEF but not Id-1-/- cells; moreover, we were able to inhibit the invasion ability of MCF7-E6/E7 and BT20-E6/E7 using Id-1 antisense retroviruses. Furthermore, we report that E6/E7 oncoproteins up-regulate Id-1 promoter activity in MCF7 and BT20 cells. We also found that HPV type 16 is present in all invasive and metastatic breast cancer and less frequently in in-situ breast cancer as opposed to normal mammary tissue. In parallel, we demonstrate that Id-1 over-expression is correlated with the presence of HPV type 16 in human invasive and metastatic breast cancer. These data suggest that high-risk HPV infections can induce cell invasion and metastasis in breast cancer through Id-1 regulation.

The Eph/Ephrin family in cancer metastasis: communication at the service of invasion

Cancer cells rely on intercellular communication throughout the different stages of their transformation and progression into metastasis. They do so by co-opting different processes such as cell-cell junctions, growth factors, receptors, and vesicular release. Initially characterized in neuronal and vascular tissues, Ephs and Ephrins, the largest family of receptor tyrosine kinases, comprised of two classes (i.e., A and B types), is increasingly scrutinized by cancer researchers. These proteins possess the particular features of both the receptors and ligands being membrane-bound which, via mandatory direct cell-cell interactions, undergo a bidirectional signal transduction initiated from both the receptor and the ligand. Following cell–cell interactions, Ephs/Ephrins behave as guidance molecules which trigger both repulsive and attractive signals, so as to direct the movement of cells through their immediate microenvironment. They also direct processes which include sorting and positioning and cytoskeleton rearrangements, thus making them perfect candidates for the control of the metastatic process. In fact, the role of Ephs and Ephrins in cancer progression has been demonstrated for many of the family members and they, surprisingly, have both tumor promoter and suppressor functions in different cellular contexts. They are also able to coordinate between multiple processes including cell survival, proliferation, differentiation, adhesion, motility, and invasion. This review is an attempt to summarize the data available on these Ephs/Ephrins’ biological functions which contribute to the onset of aggressive cancers. I will also provide an overview of the factors which could explain the functional differences demonstrated by Ephs and Ephrins at different stages of tumor progression and whose elucidation is warranted for any future therapeutic targeting of this signaling pathway in cancer metastasis.

The effect of tibolone on proliferation, differentiation and apoptosis in normal breast cells

Tissue homeostasis is the result of a balance between proliferation, differentiation and apoptosis, with apoptosis, or spontaneous programmed cell death, being thought to play a major role in the growth and regulation of both normal and tumor tissue. The expression of bcl-2protein plays an important role in controlling apoptosis, and this protein has been shown to be influenced by fluctuations in hormone levels during the menstrual cycle. Although considerable work has been carried out investigating the effect of estrogens on normal breast cells and breast cancer cell lines, less is known about the effect of progestins, and very little has been published on the tissue-specific hormone tibolone, which exhibits estrogenic, progestogenic and androgenic characteristics. Studies with tibolone, using normal human breast epithelium cells, demonstrate that this hormone displays a progestin-likeprofile and has favorable effects with respect to breast tumorigenesis. Tibolone decreases cell proliferation rates, prom...

Putative chemopreventive molecules can increase Nrf2-regulated cell defense in some human cancer cell lines, resulting in resistance to common cytotoxic therapies

Nrf2 is a key transcription factor, which induces a cytoprotective gene array. Nrf2 is regulated at the posttranslational level through proteasomal degradation through an interaction with the adapter protein Keap1. High levels of Nrf2, resulting from a loss of function mutation in Keap1, were reported in chemoresistant non-small cell lung cancer. We observed very low levels of Nrf2 and of Nrf2-regulated detoxification proteins as a frequent phenotype in the more chemosensitive breast cancer, and when engineering increased Nrf2 levels, we found resistance to both doxorubicin and paclitaxel. We here show that basal Nrf2 levels in different cell lines correlate with their respective sensitivity to a common cytotoxic chemotherapy. Nrf2 and its regulated genes and proteins are the targets of a major strategy in cancer prevention. Molecules that interfere with the Nrf2–Keap1–Cul3 protein–protein interactions result in higher levels of Nrf2. Both naturally occurring and synthetic molecules with this effect have been suggested as clinical chemopreventive agents, including molecules derived from cruciferous vegetables such as the isothiocyanate sulforaphane and even green tea polyphenols. Here, we determine the impact of these putative chemopreventive agents on the sensitivity of established cancer cell lines to chemotherapy. We confirmed that these molecules do increase Nrf2 and detoxification enzyme levels in breast cancer cell lines with very low basal Nrf2 levels, and this is associated with significant chemoresistance to cytotoxic drugs. Both effects are less in another breast cancer cell line with intermediate Nrf2, and in lung cancer cells with high Nrf2, these same molecules have no effect on Nrf2 but do actually enhance chemoresitance. While the details of dose and schedule of these agents require further study in in vivo models, these data sound a cautionary note for the use of these agents in patients with established cancers who are undergoing chemotherapy treatment.

Platelet-type 12-lipoxygenase induces MMP9 expression and cellular invasion via activation of PI3K/Akt/NF-κB.

Prostate cancer is the most frequently diagnosed cancer and the second leading cause of death in males in the United States. Using human prostate cancer specimens, the authors have previously shown that elevated expression levels of 12‐lipoxygenase (12‐LOX) occurred more frequently in advanced stage, high‐grade prostate cancer, suggesting that 12‐LOX expression is associated with carcinoma progression and invasion. Previous reports from their group and others have shown that 12‐LOX is a positive modulator of invasion and metastasis; however, the mechanism remains unclear. In this work, a new link between 12‐LOX and the matrix metalloproteinase 9 (MMP9) in prostate cancer angiogenesis is reported. This study demonstrated that overexpression of 12‐LOX in prostate cancer PC‐3 cells resulted in elevated expression of MMP9 mRNA, protein and secretion. Exogenous addition of 12(S)‐hydroxy eicosatetraenoic acid, the sole and stable end product of arachidonic acid metabolism by 12‐LOX, is able to increase MMP9 expression in wild‐type PC‐3 cells. Furthermore, using pharmacological and genetic inhibition approaches, it was found that 12‐LOX activates phosphoinositol 3 kinase (PI3K)/Akt, which results in nuclear factor‐kappa B (NF‐κB)‐driven MMP9 expression, ensuing in enhanced chemoattraction of endothelial cells. Specific inhibitors of 12‐LOX, PI3K or NF‐κB inhibited MMP9 expression in 12‐LOX‐expressing PC‐3 cells and resulted in the blockade of the migratory ability of endothelial cells. In summary, the authors have identified a new pathway by which overexpression of 12‐LOX in prostate cancer cells leads to augmented production of MMP9 via activation of PI3K/Akt/NF‐κB signaling. The role of 12‐LOX‐mediated MMP9 secretion in endothelial cell migration may account for the proangiogenic function of 12‐LOX in prostate cancer.

Platelet-type 12-lipoxygenase activates NF-κB in prostate cancer cells

Abstract Platelet-type arachidonate 12-lipoxygenase (12-LOX) is highly expressed in many types of cancers and plays an important role in cancer pathophysiology. Arachidonic acid metabolism by 12-LOX results in the stable end product 12( S )-hydroxy eicosatetraenoic acid (12( S )-HETE), which is a signaling molecule with effects on cell proliferation, motility, invasiveness, angiogenesis, and inhibition of apoptosis. The myriad biological activities manifested by 12( S )-HETE appear to be mediated, at least in part, by the activation of NF-κB. Overexpression of the 12-LOX in PC-3 prostate cancer cells resulted in the constitutive activation of the transcription factor. The enzymatic product of arachidonic acid metabolism, 12( S )-HETE, mediates the activation of NF-κB by the 12-LOX. 12( S )-HETE treatment of PC-3 cells induced the degradation of IκB by the S6 proteasomal pathway and the activated NF-κB translocated to the nucleus causing κB-induced transcription. Specificity of the NF-κB activation by 12( S )-HETE was established by the use of a 12-LOX-specific inhibitor and 13( S )-HODE, a known 12( S )-HETE antagonist. Considering the known involvement of MAP kinase pathway in NF-κB activation and that of 12( S )-HETE in MAP kinase pathway, 12-LOX present in prostate cancer tissues may contribute to the constitutive activation of NF-κB in prostate cancer cells.

The EphB2 tumor suppressor induces autophagic cell death via concomitant activation of the ERK1/2 and PI3K pathways

EphB2 is a tyrosine kinase receptor that has been shown to be a tumor suppressor gene in various cancers. However the mechanisms of this function are unknown. We report that EphB2 induces a form of cell death that does not involve the formation of apoptotic bodies or nuclear fragmentation and is instead accompanied by extensive vacuolization. Transmission electron microscopy demonstrates cytoplasmic vacuoles in EphB2-overexpressing cells that resembled autophagosomes. Using an EYFP-LC3 fusion protein and immunoblotting, we detected LC3 aggregation and conversion from form I to form II, both hallmarks of autophagy, in EphB2-transfected cells. Silencing of the autophagy regulating genes ATG5 or ATG7 using shRNAs, strongly prevented EphB2-induced cell death, further confirming its autophagic nature. EphB2 expression results in mitochondrial depolarization and translocation of cytochrome c from the mitochondria to the cytosol. Mapping of signaling pathways revealed novel information about the mechanisms of action of EphB2. We demonstrated that the MAPK pathway is important in the pro-death action of EphB2, through ERK1/2 phosphorylation and inhibition of this pathway using PD98059 counters EphB2-driven cell death. In addition, we found that inhibition of class III PI3K pathway, using the autophagy inhibitor 3MA, but not class I PI3K inhibition using LY294002, also effectively blocks EphB2-induced cell death. Finally, EphB2 expression inactivates Akt, which is a known inhibitor of autophagy. In conclusion, the EphB2 receptor induces an autophagic cell death that is mediated through the ERK1/2 and PI3K/Akt pathways.