78 research outputs found
Regulation of protein kinase C-η in breast cancer cells
No full textProtein kinase C-η (PKCη) is involved in cell proliferation, differentiation and plays an anti-apoptotic role in various cancer models. The purpose of this dissertation is to understand the regulation of PKCη in cell death signaling in breast cancer cells.
Tumor promoting phorbol esters are potent activators of PKC and prolonged treatment with these activators leads to downregulation and desensitization of PKC signaling. PKCη resists phorbol ester-induced downregulation and upregulated in response to prolonged treatment. We have found that phosphorylation of PKCη at conserved serine/threonine sites is increased by phorbol ester treatment. Single mutations that prevent phosphorylation at these conserved sites resulted in downregulation of PKCη. These results suggest that phosphorylation of PKCη at conserved sites prevents its downregulation.
We have found that PKCη can be significantly downregulated by inhibition of two pathways: PKC and phosphoinositide 3-kinase (PI3K) using pharmacological inhibitors. Inhibitors of PKC and PI3K induce dephosphorylation of PKCη by a calyculin A-specific phosphatase. These inhibitors differ in their ability to degrade PKCη via the proteasome-mediated pathway. Downregulation of PKCη by PKC inhibitor is not mediated by the proteasome degradation pathway, whereas downregulation by PI3K inhibitor is dependent on the proteasome. These results demonstrate that dephosphorylation is important for PKCη downregulation and this occurs through two distinct mechanisms.
A comparison of PKC protein levels in a series of isogenic cell lines representing a breast cancer progression model revealed that PKCη protein levels increased from the normal, benign stage to the pre-malignant and metastatic stages. These results indicate that PKCη plays a potential role in breast cancer progression. Further analysis indicated that overexpression of PKCη in the MCF-7 breast adenocarcinoma cell line conferred resistance to tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL)-induced cell death. Therefore, PKCη protects breast cancer cells from TRAIL-induced apoptosis. Depletion of PKCη by small interfering RNA (siRNA) resulted in increased dimerization of pro-apoptotic Bax and decreased induction of anti-apoptotic Mcl-1 by TRAIL. These results indicate that PKCη may exert its anti-apoptotic effect by targeting Bcl-2 proteins, Bax and Mcl-1
Transcriptional regulation of the human RLIP76 gene
No full textA 76-kDa Ral-interacting protein (RLIP76) has been implicated in the pathogenesis of cancer and diabetes. It is often overexpressed in human malignant cell lines and human tumor samples and has been associated with metastasis and chemoresistance. RLIP76 homozygous knockout mice exhibit increased insulin sensitivity, hypoglycemia, and hypolipidemia, and resist cancer development. Little is known about the mechanism by which the expression of RLIP76 is regulated. In the present study, we functionally characterized the RLIP76 promoter using deletion mapping and mutational analysis to investigate the regulation of RLIP76 transcription. We have identified the promoter regions important for RLIP76 transcription, including a strong positive cis-acting element (-167 to -152) in the proximal promoter containing overlapping consensus cMYB and cETS binding sites. Transcription factor cMYB and the coactivator p300 associate with the RLIP76 gene promoter as shown by CHIP assay. Knockdown of p300 in cells reduces the activity of the promoter fragment containing wild type cMYB/cETS binding site in comparison to that with deleted or mutated cMYB/cETS binding site. Knockdown of p300 also decreases the RLIP76 expression as indicated by immunoblotting, immunocytochemistry and flow cytometry analysis. Thus, we report for the first time that p300 associates with the RLIP76 promoter via overlapping cMYB and cETS binding sites and regulates RLIP76 promoter activity and expression in HEK293 and MCF7 cells
Protein Kinase C-eta Signalling in Breast Cancer
No full textPal, Deepanwita, Protein kinase C-eta signaling in breast cancer. Doctor of Philosophy (Biochemistry and Molecular Biology), November, 2013, 117 pp, 14 illustrations, 260 References Protein kinase C-eta (PKCη) is a novel member of the PKC family that is important for several cellular processes. PKCη is overexpressed in breast cancer and has been associated with chemotherapeutic resistance. PKCη is the only phorbol ester-sensitive PKC isozyme that resists downregulation upon prolonged treatment with tumorpromoting phorbol esters suggesting its unique regulation. The purpose of this dissertation is to elucidate the mechanism of PKCη regulation and its functional relevance in breast cancer. We have shown that PKCη is upregulated by several structurally and functionally distinct PKC activators in contrast to other PKC isozymes. Activator-induced upregulation of PKCη was associated with its phosphorylation. Our results indicate that novel PKCs are involved in the upregulation of PKCη by PKC activators. We also made a novel observation that PKCη is downregulated via two distinct mechanisms. While inhibition of PKC caused the downregulation of PKCη via proteasome-independent pathway, inhibition of PDK1 led to PKCη downregulation via proteasome-dependent pathway. We further demonstrated that PKCη is important for the growth and survival of breast cancer cells. The unique regulation of PKCη and its implications on breast cancer growth and survival suggests that this pathway could be selectively exploited for targeted therapies for breast cancer
Dissecting the Role of Protein Kinase C-Epsilon in Breast Cancer
No full textProtein kinase C-epsilon (PKCε) has pro-tumor functions in many cancers including breast cancer. The purpose of this dissertation is to understand the role of PKCε in fundamental processes that are associated with breast cancer development and progression.
PKCε is known to promote the survival of breast cancer cells. Autophagy is a process of cellular self-digestion that can mediate cell survival during stress. We have found that PKCε overexpression increases the basal autophagy in breast cancer cells while its depletion reduces it. Moreover, the effect of PKCε on autophagy is isozyme specific. Regulation by PKCε is not limited to basal autophagy as it also mediated starvation-induced autophagy. Looking for the possible mechanisms, we found that PKCε negatively regulates mammalian target of rapamycin (mTOR), which is the master regulator of autophagy. These results show that PKCε positively regulates autophagy, likely, via inhibition of mTOR.
PKCε overexpression in mammary epithelial cells led to morphological changes indicating its role in regulation of cell plasticity. Further analysis revealed that PKCε promotes epithelial to mesenchymal transition (EMT), which is an early step in cancer metastasis. In addition, PKCε mediated transforming growth factor-beta (TGFβ)-induced EMT partially via Snail, which is a crucial EMT effector. Moreover, PKCε promoted cell migration and anoikis Ii resistance which are hallmarks of EMT. To examine the phenotypic effect of PKCε manipulation in a physiologically relevant context, we employed three dimensional (3D) cell culture model. We found that PKCε overexpression led to disruption of acinar morphogenesis in 3D culture. These results indicate a causal role for PKCε in breast tumor development and progressio
Involvement of S6 Kinase in Breast Cancer
No full textSridharan, S., Involvement of S6 Kinase in Breast Cancer. Doctor of Philosophy (Cancer Biology), November 2013, 129pp, 19 illustrations, 215 references. The 40S ribosomal protein S6 Kinase (S6K) is activated downstream of the mammalian target of rapamycin (mTOR)and is believed to play and important role in protein translation. In mammalian cells S6K is represented by two highly homologous proteins, S6K1 and S6K2. Both homologs have been shown to be amplicfied and over expressed in breast cancer cells and tissues. While the regulation and functions of S6K1 have been addressed, little is known about those of S6K2 . Hence we sought to examine the causes and consequences of elevated S6K2 levels in breast cancer cells. While the depletion of S6K1 decreased breast cancer cell death, silencing of S6K2 substantially increased it in response to apoptotic and chemotherapeutic agents. We then explored the mechanism by which S6K2 mediates survival and observed that in contrast to S6K1, S6K2 depletion decreased the activation of the prosurvival protein Akt and increased the level of proapoptotic proteins p53 and bid. Following this observation, we sought to determine the pathways(s) contributing to the overexpression of S6K2 in breast cancer cells. Due to its role as a prognostic indicator in estrogen receptor (ER) – positive tumors, we studied the role of the estrogen signaling pathways in regulating S6K2 levels. Estradiol and estrogen receptor alpha (ERα) positively regulated S6K2 protein but did not affect its mRNA levels, suggesting post-transcriptional regulation. We further observed that S6K2 regulated cell survival downstream of estrogen in ER-positive breast cancer cells. These findings strongly suggest that S6K2 is critical for the survival of breast cancer cells and that targeting S6K2 in combination with chemotherapeutic agents is a novel strategy to promote breast cancer cell death
Regulation of Autophagy by Protein Kinase C-ε in Breast Cancer Cells
No full textProtein kinase C-ε (PKCε), an anti-apoptotic protein, plays critical roles in breast cancer development and progression. Although autophagy is an important survival mechanism, it is not known if PKCε regulates autophagy in breast cancer cells. We have shown that silencing of PKCε by siRNA inhibited basal and starvation-induced autophagy in T47D breast cancer cells as determined by the decrease in LC3-II, increase in p62, and decrease in autophagy puncta both in the presence and absence of bafilomycin A1. The mechanistic target of rapamycin (mTOR) associates with Raptor or Rictor to form complex-1 (mTORC1) or complex-2 (mTORC2), respectively. Knockdown of PKCε attenuated an increase in autophagy caused by the depletion of Raptor and Rictor. Overexpression of PKCε in MCF-7 cells caused activation of mTORC1 and an increase in LC3-I, LC3-II, and p62. The mTORC1 inhibitor rapamycin abolished the increase in LC3-I and p62. Knockdown of mTOR and Rictor or starvation enhanced autophagy in PKCε overexpressing cells. While overexpression of PKCε in MCF-7 cells inhibited apoptosis, it induced autophagy in response to tumor necrosis factor-α. However, inhibition of autophagy by Atg5 knockdown restored apoptosis in PKCε-overexpressing cells. Thus, PKCε promotes breast cancer cell survival not only by inhibiting apoptosis but also by inducing autophagy
The Enigmatic Protein Kinase C-eta
No full textThe purpose of this review article is to discuss how PKCη regulates various cellular processes that may contribute to its contrasting roles in cancer. Protein kinase C-eta (PKCη) is a unique member of the PKC family since its regulation is distinct from other PKC isozymes. PKCη was shown to regulate cell proliferation, differentiation and cell death
Comparative study of the physical symptoms following bilateral mastectomy with immediate breast reconstruction and delayed breast reconstruction
No full textBreast reconstruction following a mastectomy can be done either immediately, at the time of the mastectomy, or following a recovery from the original surgery. This study compares the post-operative symptom experiences of these two groups of patients through secondary data analysis, using an existing survey, “How Women Decide” conducted at Texas Health Harris Methodist Hospital, Fort Worth (THFW). Statistical analysis was performed using SPSS 20. Tiredness/fatigue is the only symptoms which approached significance between the two groups; patients in the immediate breast reconstruction group experienced more tiredness/fatigue. No other aspects covered in the survey differed between the two groups. The study also found that hot flashes and tiredness/fatigue are the most common symptoms affecting the patients following bilateral mastectomy with breast reconstruction surgery. A larger sample size may have uncovered additional significant differences
The Regulation of P53 by Protein Kinase C in Anticancer Drug-Induced Apoptosis
No full textJohnson, C., The regulation of p53 by protein kinase C in anticancer drug-induced apoptosis. Master of Science (Microbiology and Immunology), December, 2001. 43 pp., 11 figures, references, 6 titles. The tumor suppression protein p53 has been implicated in DNA damage-induced apoptosis. Previous studies demonstrated that the protein kinase C (PKC) signal transduction pathway regulates apoptosis induced by the DNA damaging agent cisplatin and is deregulated in cisplatin-resistant cells. The present study examined whether PKC influences p53 and, hence, cellular sensitivity to cisplatin. Basal p53 levels were elevated in cisplatin-resistant HeLa (HeLa/CP) cells as compared to parental HeLa cells. Cisplatin further increased p53 levels in HeLA/CP, but not in HeLA cells. However, rottlerin, a PKC-δ inhibitor that prevents cisplatin-induced apoptosis, caused p53 accumulation in HeLa cells treated with cisplatin. Rottlerin stabilized p53 in response to cisplatin in HeLa cells, whereas cisplatin alone was sufficient to stabilize p53 in HeLa/CP cells
Effect of CRISPR MIEN1 knockout in metastatic breast cancer cells
No full textMigration and Invasion Enhancer 1 (MIEN1) is an oncogene which is involved in facilitating the migration and invasion of cancer cells through actin dynamics and gene expression. Increased MIEN1 expression in many types of tumors correlates with disease progression and metastatic propensity. The precise mechanism by which MIEN1 functions is yet to be understood. The goal of these studies is to progress toward determination of the mechanisms and genetic context in which MIEN1 functions contribute to cancer progression. It was hypothesized that Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) mediated knockout of MIEN1 in metastatic breast cancer cells would result in reduced migration and invasion. CRISPR genome editing effectively produced specific genomic deletions in the MIEN1 gene which led to the elimination of its expression in these breast cancer cells. Migration in MDA-MB-231 (231) MIEN1 knockout (MIEN1-KO) cells exhibited no difference when compared to parental 231, which was in contrast with previous siRNA studies. Signaling in several MIEN1-KO pools was inconsistent. Knocking out MIEN1 in 231 derivative cell lines showed few significant alterations in the growth, migration, invasion, signaling, despite significant changes in metabolism. However, re-expression of the MIEN1 protein containing a mutant immunoreceptor tyrosine-based activation motif (ITAM) domain resulted in significantly decreased invasion. This revealed that MIEN1-KO 231 derivative cells were susceptible to interference of compensatory mechanisms and demonstrates the importance of the migration and invasion pathways in which MIEN1 participates in breast cancer metastasis. These findings also suggest MIEN1 may still be a promising therapeutic target to inhibit metastasis if inhibitors can be developed which block ITAM function without affecting localization or expression
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