Cancer Research Cancer Medicine 8 Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 69, 65, January 1, 2009. doi: 10.1158/0008-5472.CAN-08-0377
© 2009 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cohen, E. E.W.
Right arrow Articles by Rosner, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cohen, E. E.W.
Right arrow Articles by Rosner, M. R.

Cell, Tumor, and Stem Cell Biology

A Feed-Forward Loop Involving Protein Kinase C{alpha} and MicroRNAs Regulates Tumor Cell Cycle

Ezra E.W. Cohen1, Hongyan Zhu2, Mark W. Lingen1,3, Leslie E. Martin3, Wen-Liang Kuo1, Eugene A. Choi4, Masha Kocherginsky5, Joel S. Parker6, Christine H. Chung7 and Marsha Rich Rosner2

1 Section of Hematology/Oncology, Department of Medicine, 2 Ben May Department for Cancer Research, 3 Department of Pathology and Department of Radiation and Cellular Oncology, 4 Section of General Surgery, Department of Surgery, and 5 Department of Health Studies, University of Chicago, Chicago, Illinois; 6 Expression Analysis, Durham, North Carolina; and 7 Divison of Hematology/Oncology, Department of Medicine and Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee

Requests for reprints: Marsha Rosner, Ben May Department for Cancer Research, Gordon Center for Integrative Sciences, University of Chicago, 929 East 57th Street, W421C, Chicago, IL 60637. Phone: 312-702-0380; E-mail: m-rosner{at}uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Protein kinase C{alpha} (PKC{alpha}) has been implicated in cancer, but the mechanism is largely unknown. Here, we show that PKC{alpha} promotes head and neck squamous cell carcinoma (SCCHN) by a feed-forward network leading to cell cycle deregulation. PKC{alpha} inhibitors decrease proliferation in SCCHN cell lines and xenografted tumors. PKC{alpha} inhibition or depletion in tumor cells decreases DNA synthesis by suppressing extracellular signal-regulated kinase phosphorylation and cyclin E synthesis. Additionally, PKC{alpha} down-regulates miR-15a, a microRNA that directly inhibits protein synthesis of cyclin E, as well as other cell cycle regulators. Furthermore, both PKC{alpha} and cyclin E protein expression are increased in primary tumors, and PKC{alpha} inversely correlates with miR-15a expression in primary tumors. Finally, PKC{alpha} is associated with poor prognosis in SCCHN. These results identify PKC{alpha} as a key regulator of SCCHN tumor cell growth by a mechanism involving activation of mitogen-activated protein kinase, an initiator of the cell cycle, and suppression of miR-15a, an inhibitor of DNA synthesis. Although the specific components may be different, this type of feed-forward loop network, consisting of a stimulus that activates a positive signal and removes a negative brake, is likely to be a general one that enables induction of DNA synthesis by a variety of growth or oncogenic stimuli. [Cancer Res 2009;69(1):65–74]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The protein kinase C (PKC) serine/threonine kinases [classical ({alpha}, β, and {gamma}), novel ({delta}, {varepsilon}, {eta}, and {theta}), and atypical ({zeta} and {iota}/{lambda})] are critical mediators of a multitude of cell signaling events (1). Despite their integral participation in cellular physiology, relatively little is known regarding their expression and function in malignant disease, such as squamous cell carcinoma of head and neck (SCCHN).

PKC{alpha} and PKC{zeta} are among the isoforms expressed in normal adult human keratinocytes and SCCHN (2). We have previously shown that PKC{zeta} activation is required in epidermal growth factor (EGF)–stimulated mitogen-activated protein kinase (MAPK) signaling and proliferation in SCCHN cell lines, and its expression increases with tumor progression in SCCHN tissues (2). PKC{alpha} is implicated in the progression of other epithelial-derived tumors, including breast and lung (3), and is associated with poor prognosis in hepatocellular carcinoma (4). Alterations in keratinocyte differentiation markers caused by oncogenic Ras and characteristic of late-stage papilloma development are also mediated by PKC{alpha} (5). Although late-stage keratinocyte differentiation markers are induced by PKC{alpha} and oncogenic Ras, the cells do not undergo the normal apoptotic processes characteristic of envelope cornification (5). These data suggest that PKC{alpha} in the context of an active oncogene does not effectively promote terminal keratinocyte differentiation. Consistent with these results, a broad spectrum PKC inhibitor that is most potent against classic and novel isoforms, chelerythrine chloride, reduced tumor growth in a xenograft model of SCCHN (6). These studies suggest that PKC{alpha} plays a key role in the growth of SCCHN.

The cell cycle is a tightly controlled process that involves transient expression of specific cyclins in association with cyclin-dependent kinases (cdk), leading to transcription factor activation (7). The G1-S phase transition requires initial expression of cyclin D complexed with cdk4/6 followed by induction of cyclin E complexed with cdk2; each cyclin-activated kinase phosphorylates Rb, releasing E2F1-3 transcription factors that promote DNA synthesis.

MicroRNAs (miRNA), 21 to 23 nucleotide RNAs that regulate the stability or translational efficiency of target mRNAs, have been implicated in diverse cellular processes relevant to cancer, including cell cycle (8, 9). Specific miRs, including miR-17-5p and miR-20a, regulate E2F family members (28–14). The miR-16-1 family, including miR-15a, affects cell cycle progression (11).

In the present study, we show that PKC{alpha} controls DNA synthesis via a feed-forward network involving miR-15a regulation of cyclin E. Tissue array analysis of PKC{alpha} and cyclin E protein expression revealed a progressive increase from normal oral mucosa and dysplasia to SCCHN. Finally, in a cohort of patients with SCCHN, higher PKC{alpha} gene expression was significantly associated with lower miR-15a levels and adverse outcome, highlighting the relevance of this signaling cascade.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Immunohistochemistry. Immunohistochemistry was performed as previously described and scored on a 0 to 3+ scale (2). Specific antibodies used were PKC{alpha} rabbit primary antibody (Santa Cruz Biotechnology), phosphorylated PKC{alpha} rabbit primary antibody (Cell Signaling), and PKCβ mouse primary antibody (Sigma).

Growth of human tumor xenografts. SQ20B tumor cells were injected into the right hind limbs of female athymic nude mice as described (6).

DNA microarray analyses. Biotin-labeled RNA was hybridized onto Affymetrix Human Genome U133 Plus 2.0 GeneChip. Raw data containing ~55,000 Affymetrix probe sets (representing 31,000 genes) were normalized, and statistical significance was determined using significance analyses of microarrays (false discovery rate, <28%), as previously described (15, 16).

RNA isolation and real-time PCR from human tissue. All tissue samples, collected under a Vanderbilt University Institutional Review Board–approved protocol, were macrodissected to obtain at least 70% tumor cells (17). The RNA was purified using PureLink Total RNA Purification System and miRNA Isolation kit (Invitrogen). miR-15a quantitative reverse transcription–PCR (qRT-PCR) used TaqMan miRNA assays (ABI) normalized to U6 CT values.

Additional procedures are described in Supplementary Materials and Methods.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Inhibition of PKC{alpha} reduces DNA synthesis and cell growth in multiple SCCHN cell lines in vitro and tumor growth in vivo. Because PKC{alpha} is expressed in all SCCHN cell lines examined (2), we determined whether PKC{alpha} is required for DNA synthesis. Gö6976 is an inhibitor of the classic PKC isoforms ({alpha}, β, {gamma}) with a reported IC50 of 280 nmol/L (18). Because SCCHN cell lines do not express the β and {gamma} PKC isoforms (2), this inhibitor primarily targets PKC{alpha} in these cells. Serum-starved SCC61, CAL27, HN31, and SQ20B cells were pretreated with 280 nmol/L Gö6976 for 24 hours before serum-induced BrdUrd incorporation into DNA. Dose-response analysis showed 50 to 280 nmol/L Gö6976 inhibited SQ20B DNA synthesis and 280 nmol/L Gö6976 suppressed DNA synthesis in all cell lines (Fig. 1A ). Time-course analysis revealed complete inhibition of SQ20B DNA synthesis by 9 to 12 hours of Gö6976 treatment. Fluorescence-activated cell sorting (FACS) analysis of SQ20B cells confirmed that Gö6976-treated cells were arrested in S phase (Supplementary Fig. S1A). To confirm that PKC{alpha} is required for DNA synthesis, two cell lines (SCC61 and SQ20B) were transfected with either control or PKC{alpha} small interfering RNA (siRNA) before serum-stimulated BrdUrd incorporation (Fig. 1B). Protein immunoblotting confirmed siRNA specificity for PKC{alpha} (Supplementary Fig. S1B). These results indicate that PKC{alpha} is required for DNA synthesis in SCCHN tumor cells.


Figure 1
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. PKC{alpha} inhibition reduces DNA synthesis, cell viability, and tumor growth in vivo. A, SCC61, SQ20B, CAL27, and HN31 cell lines were incubated in serum-free media (control), serum alone (serum), or serum with 280 nmol/L of Gö6976 (Gö 280 nmol/L) for up to 24 h and DNA synthesis was quantitated for BrdUrd incorporation, as described in Materials and Methods. B, SCC61 (left) and SQ20B (right) cells were transfected with control or PKC{alpha} siRNA. DNA synthesis was quantitated for BrdUrd incorporation, as described in Materials and Methods. All BrdUrd incorporation results are the mean ± SE of three independent experiments. Immunoblotting for PKC{alpha} and actin was performed in conjunction with BrdUrd incorporation after control and PKC{alpha} siRNA transfection, as described in Materials and Methods. C, SCC61 (left) and SQ20B (right) cells were transfected with control or PKC{alpha} siRNA, and cell survival was quantitated by MTT assay, as described in Materials and Methods. All MTT results are the mean ± SE of three independent experiments. D, SQ20B tumor xenografts were established in female athymic nude mice. After reaching an average size of 150 mm3, the mice (n = 5) were treated with 0.1% DMSO in PBS (control) or Gö6976 (n = 28 per group) at 0.06 mg/kg/wk or 0.12 mg/kg/wk by i.v. tail vein injection. Points, mean tumor size; bars, SE.

 
To assess whether inhibition of PKC{alpha} decreases cell growth in SCC61 and SQ20B cells transfected with control or PKC{alpha} siRNA, we used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay that measures changes in metabolism. The inhibition was generally less robust than for DNA synthesis, possibly due to differences in kinetics or assays, but in both cell lines, PKC{alpha} depletion reduced cell growth (Fig. 1C). Pretreatment with Gö6976 reduced proliferating SCC61, CAL27, HN31, and SQ20B cells in a dose-dependent manner with almost a complete inhibition at 100 nmol/L (Supplementary Fig. S1C). These results show that PKC{alpha} is a critical mediator of SCCHN cell proliferation.

To determine whether PKC{alpha} inhibitors affect SCCHN tumor growth in vivo, hind limb SQ20B tumor xenografts were established in female athymic nude mice. Administration of Gö6976 dramatically retarded growth of the SQ20B xenografts (Fig. 1D). Although we cannot rule out effects on other cells or drug targets within the microenvironment, these results are consistent with the inhibition of SCCHN tumor cell growth in culture by PKC{alpha}.

PKC{alpha} inhibition suppresses genes and proteins that regulate the cell cycle. Previous studies have shown that PKC{alpha} and other PKC isoforms, such as PKC{zeta}, stimulate the MAPK signaling cascade via Raf activation (2, 19, 20), and MAPK activation is required for DNA synthesis in SQ20B cells (2). Consistent with this observation, cell stimulation with serum or a PKC inducer, phorbol 12,1h3-dibutylate (PDBu), activates extracellular signal-regulated kinase (ERK) and PKC{alpha} siRNA decreases ERK activation (Fig. 2A ). PKC{alpha} siRNA also reduces baseline ERK activation in PDBu and serum-treated SQ20B cells. Thus, under these conditions, PKC{alpha} is both necessary and sufficient for MAPK signaling.


Figure 2
View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. PKC{alpha} inhibition induces changes in MAPK activity and regulates cell cycle genes. A, decreased ERK activity in SQ20B cells with PKC{alpha} inhibition. SQ20B cells were transfected with control (siControl) or PKC{alpha} (siPKC{alpha}) siRNA. After 48 h in serum-free media, cells were incubated with increasing concentrations of PDBu (left) or serum (right) for 30 min. Whole-cell lysates were probed for phosphorylated and total ERK, and blots were quantified, as described in Materials and Methods. Graphs represent quantification of the ratio of phosphorylated ERK to total ERK protein. Representative experiments are shown for at least two independent experiments. B, gene array heat map of cell cycle genes in the SQ20B cell line with decreased expression at 24 h upon exposure to 100 nmol/L Gö6976 (left). Right, RT-PCR expression of selected cell cycle genes at 0 h (black columns), 12 h (gray columns), and 24 h (white columns) shown as fold change normalized to untreated control. C, protein immunoblotting and immunoprecipitation of selected cell cycle genes upon exposure to 100 nmol/L Gö6976 at 0, 12, and 24 h. SQ20B cells were serum starved for 48 h, followed by treatment in serum with 100 nmol/L Gö6976 for 0, 12, and 24 h. Total protein was extracted from cells and immunoblotted for cyclin E, E2F2, cyclin A, MCM6, and {gamma}-tubulin, as described in Materials and Methods (left). Cell lysates were immunoprecipitated with an anti-Cdk2 antibody, and the immunoprecipitates were examined by immunoblotting to detect cdk2-complexed cyclin E (cdk2 IP/cyclin E IB; right). Total protein lysates were also probed for cyclin E and cdk2 antibodies. Representative data from three separate experiments. D, E2F 1-3 protein levels in SQ20B cells. SQ20B cells were serum-starved for 48 h, followed by treatment in serum with 100 nmol/L Gö6976 for 0, 12, and 24 h. Total protein lysates were probed with the respective antibodies. Lysate from untreated U2OS cells was used as a positive control for E2F1.

 
Although MAPK activation initiates tumor cell proliferation, additional mechanisms sustain growth response. To identify potential effectors of PKC{alpha} that regulate DNA synthesis in SCCHN cells, we inhibited PKC{alpha} and analyzed changes in gene expression. Serum-starved SQ20B cells were treated with 100 nmol/L Gö6976 for 0, 12, or 24 hours, and gene expression changes were determined by DNA microarrays. To detect differential expression of low abundance regulatory genes, data were queried using 171 probes related to cell cycle regulation (21). Fifty-five probes (39 unique genes) were differentially expressed upon comparison of the 0 versus 24 hour data sets (Supplementary Fig. S2). Eight of the 39 genes had decreased expression between 12 and 24 hours after Gö6976 treatment (Fig. 2B, left).

Inhibition of four genes (cyclin E1, E2F2, MCM6, and PCNA) was confirmed by qRT-PCR (Fig. 2B, right). Among E2F2-regulated genes are cyclins E1 and E2, E2F2 itself, PCNA (22, 23), and the MCM proteins (24). Cyclins E1 and E2 interact with cdk2 to form a serine/threonine kinase holoenzyme complex that phosphorylates the Rb protein, relieving repression of E2F2-mediated transcription (7). MCMs, minichromosome maintenance proteins, are DNA helicases required for replication (24), and PCNA, a DNA polymerase cofactor, recruits key factors to the replication fork (25).

Analysis of cyclin E and E2F2 protein levels by immunoblotting similarly revealed a decrease in response to the PKC{alpha} inhibitor. Cell treatment with 100 nmol/L Gö6976 for 0, 12, and 24 hours significantly reduced cyclin E and E2F2 protein levels (Fig. 2C). No loss of cyclin E before 10-hour treatment, consistent with the DNA synthesis results, was observed (data not shown). In contrast, no change in MCM6, cdk2, or cyclin A protein levels was detected. The cell cycle transition to S phase requires phosphorylation by cyclin E complexed with cdk2. Immunoprecipitation of cdk2 revealed a dramatic decrease in cyclin E associated with cdk2 after Gö6976 treatment, indicative of cdk2 kinase inactivation (Fig. 2C). Although PCNA was similarly regulated, expression levels for the other E2F family members (1 and 3) were not significantly altered by PKC{alpha} inhibition (Fig. 2D). These results suggest that lowered cyclin E and, possibly, E2F2 protein expression caused by PKC{alpha} inhibition accounts for the decrease in DNA synthesis.

We then tested whether cyclin E or E2F2 are required for DNA synthesis in SCCHN cells. SQ20B cells were depleted of human cyclin E by transfection with siRNA (Fig. 3A ). Analysis of BrdUrd incorporation into DNA indicates that cyclin E expression is required for DNA synthesis. Conversely, expression of cyclin E was sufficient to significantly rescue BrdUrd incorporation into DNA after 100 nmol/L Gö6976 treatment (Fig. 3B). E2F2 was also required for DNA synthesis, as shown by siRNA depletion (Fig. 3A). However, in contrast to Gö6976 treatment, PKC{alpha} depletion did not significantly decrease E2F2 protein levels. These differences between drug and siRNA effects could reflect differences in kinetics or mechanism of PKC{alpha} inactivation or, alternatively, a role for other PKC isoforms or enzymes in mediating the drug's effects on E2F2. Furthermore, E2F2 overexpression only induced a limited recovery of DNA synthesis in Gö6976-treated cells (Fig. 3B) consistent with the presence of other E2Fs in the cell. Finally, these results confirm that siRNA depletion of PKC{alpha} leads not only to inhibition of DNA synthesis but also to loss of cyclin E expression (Fig. 3A). Thus, PKC{alpha} induces a cyclin E expression that is necessary for SCCHN cell cycle progression; furthermore, cyclin E is sufficient to overcome the cell cycle block generated by loss of PKC{alpha} activity.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Cyclin E mediates PKC{alpha} induction of DNA synthesis in SQ20B cells. A, effect of siRNA on DNA synthesis. SQ20B cells were transfected with control, PKC{alpha}, cyclin E, or E2F2 siRNA, and protein expression was determined, as described in Materials and Methods (left). SQ20B cells were incubated in serum-free media (starved), serum alone (serum), serum with control siRNA, serum with 100 nmol/L Gö6976, or serum with respective siRNAs for 24 h (right). DNA synthesis was quantitated for BrdUrd incorporation, as described in Materials and Methods, and expressed as fold change compared with serum alone. B, rescue of DNA synthesis with cyclin E and E2F2 expression in Gö6976-treated cells. SQ20B cells were mock transfected or transfected with cyclin E and E2F2 expression vectors, and protein expression was determined, as described in Materials and Methods (left). SQ20B cells were incubated in serum-free media (starved), serum alone (serum), serum with Gö6976 (Gö6976), or serum with 100 nmol/L Gö6976 and the respective transfection vector(s) for 24 h. DNA synthesis was quantitated for BrdUrd incorporation, as described in Materials and Methods, and expressed as fold change compared with serum alone (right). All BrdUrd incorporation results are the mean ± SE of three independent experiments. C, change in rate of cyclin E protein synthesis. SQ20B cells were either untreated or pretreated with Gö6976 (100 nmol/L) for 12 h and then exposed to MG132 (30 µmol/L) for the times indicated. Graph represents quantification of cyclin E levels from the protein immunoblot. Cyclin E values were normalized to {alpha}-tubulin levels. Results are representative of three independent experiments.

 
PKC{alpha} regulation of cyclin E protein levels results from changes in protein synthesis or degradation. To test the former possibility, SQ20B cells were left untreated or pretreated with the PKC{alpha} inhibitor Gö6976 for 12 hours followed by addition of MG132, a proteosome inhibitor, to prevent proteolysis. Although the cyclin E level eventually plateaus due to resumed degradation, inhibition of PKC{alpha} significantly decreased the initial rate of cyclin E protein synthesis relative to that in untreated tumor cells (Fig. 3C). To test whether the proteosome is a target of PKC{alpha}, SQ20B cells were pretreated with Gö6976 and then exposed to cyclohexamide to block protein synthesis. However, no consistent increase in the rate of cyclin E degradation was observed (data not shown). These results indicate that inhibition of PKC{alpha} suppresses cyclin E protein synthesis.

PKC{alpha} regulates cyclin E synthesis through miR-15a. Our previous results established that PKC{alpha} increased DNA synthesis via enhancement of cyclin E protein levels. Both gene expression arrays and qRT-PCR analyses showed decreases in mRNA levels between 12 and 24 hours of treatment by Gö6976 (Fig. 2B). However, time-course experiments reveal that inhibition of DNA synthesis was maximal at 12 hours (Fig. 1A). In addition, our results confirm that PKC{alpha} inhibition caused maximal suppression of cyclin E protein expression by 12 hours, before subsequent changes in transcription (Fig. 2B and C). Thus, PKC{alpha} regulation of transcription rates cannot account for the kinetics of cyclin E protein enhancement.

A recently described mechanism for regulating protein synthesis involves inhibition of mRNA translation by miRs (8). To investigate whether cyclin E protein loss in response to PKC{alpha} inhibition results from induction of miRs, we searched the PicTar database8 and found that miR-15a could theoretically bind to two regions within the 3' untranslated region (3'-UTR) of the cyclin E mRNA (Fig. 4B ). qRT-PCR confirmed that PKC{alpha} inhibition by Gö6976 treatment or PKC{alpha} depletion by siRNA significantly increased miR-15a expression (Fig. 4A). The kinetics of inhibition are different because drug inhibition of kinase activity occurs more rapidly than elimination of PKC{alpha} expression. Similarly, a different siRNA that had slower kinetics for PKC{alpha} depletion required a consistent shift in the kinetics to induce miR-15a expression by 2-fold (Supplementary Fig. S3).


Figure 4
View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. PKC{alpha} regulates cyclin E expression and DNA synthesis through miR-15a. A, detection of mature miR-15a expression in SQ20B cells by quantitative real-time PCR. SQ20B cells were untreated or treated with Gö6976 (100 nmol/L, top) or PKC{alpha} siRNA (bottom) for 12 and 24 h. After miRNA extraction, qRT-PCR was performed in triplicate, as described in Materials and Methods. Columns, represent relative miRNA levels normalized to no treatment; bars, SE. B, miR-15 and PKC{alpha} regulate protein synthesis via miR-15 binding sites in the 3'-UTR of cyclin E. SQ20B cells were transfected with a luciferase reporter expressed in conjunction with control vector (Con), wild-type (Wt) 3'-UTR of cyclin E, or doubly mutated (Mut) 3'-UTR of cyclin E. The reporter-expressing cells were either transfected with pre–miR-15a or control vector (middle) or pretreated with Gö6976 (100 nmol/L) for 24 h (lower). Top, sites of miR-15a binding in the 3'-UTR of cyclin E and the mutations that were introduced. P values were determined using two-tailed Student's t test. C, effect of pre–miR-15a and anti–miR-15a on cyclin E levels in the absence or presence of Gö6976. SQ20B cells were transfected with 20 nmol/L of pre–miR-15a (Pre), 50 nmol/L of anti-miR-15a (anti), or control precursor (C) and treated (+) or untreated (–) with Gö6976 (100 nmol/L) for 24 h. Cyclin E level was determined by protein immunoblotting. Each blot of paired lanes represents an independent experiment and was not performed simultaneously with the others. D, effect of miR-15a and anti–miR-15a on DNA synthesis. Top, SQ20B cells were transfected with either 20 nmol/L of control precursor or pre-miR-15a RNA by electroporation. Cells were grown in serum-free medium (control) or serum-containing medium (all others) and exposed to Gö6976 (100 nmol/L), pre–miR-15a miRNA (pre15a), or both. BrdUrd incorporation was quantified and expressed as fold change normalized to serum. Bottom, SQ20B cells were transfected with 20 or 50 nmol/L of anti–miR-15a or Gö6976 (100 nmol/L) and grown in serum-containing medium. BrdUrd incorporation assays were performed, and levels were expressed as fold change normalized to serum. All BrdUrd incorporation results are the mean ± SE of three independent experiments.

 
We therefore determined whether miR-15a directly inhibits cyclin E translation. The 3'-UTR region of the human cyclin E1 gene containing intact (Wt) or doubly mutated miR-15a binding sites (Mut) was cloned into Renilla luciferase reporter vectors. SQ20B cells were transfected with control (Con) or cyclin E 3'-UTR–containing luciferase vectors and cotransfected with either control or miR-15a precursor microRNA (con or pre–miR-15a). Treatment with pre–miR-15a reduced cyclin E1–dependent luciferase activity in cells transfected with the Wt cyclin E 3'-UTR (Fig. 4B). By contrast, no reduction was observed with the Mut cyclin E 3' UTR, confirming that miR-15a directly inhibits cyclin E1 translation via binding to its 3'-UTR at the predicted sites. Moreover, treatment with Gö6976 further reduced luciferase activity in cells transfected with the Wt but not the Mut cyclin E 3'-UTR reporters (Fig. 4B), confirming that regulation of cyclin E1 by its 3'-UTR is both PKC{alpha}-dependent and miR-15a–dependent.

These 3'-UTR results suggest that miR-15a inhibits cyclin E protein expression. Transfection with pre–miR-15a confirmed that miR-15a reduces cyclin E protein levels in SQ20B cells; this decrease occurred both in cells that were untreated or pretreated with the PKC{alpha} inhibitor Gö6976 (Fig. 4C). Furthermore, expression of an inhibitor of miR-15a, anti-miR-15a, increases cyclin E protein expression and partially rescues the effects of Gö6976 (Fig. 4C).

Similar results were obtained for regulation of DNA synthesis by miR-15a. Transfection of pre–miR-15a into SQ20B cells inhibits DNA synthesis to a similar extent as Gö6976 (Fig. 4D). Conversely, anti–miR-15a enhances DNA synthesis and antagonizes the antiproliferative effect of Gö6976 (Fig. 4D). Consistent with an miRNA-dependent mechanism, washout experiments showed that the effects of Gö6976 treatment on cyclin E protein levels are reversible (data not shown). These results show that miR-15a is suppressed by PKC{alpha} and inhibits cyclin E expression and DNA synthesis.

Enhanced PKC{alpha} and cyclin E expression in malignant oral epithelium. If PKC{alpha} is a critical mediator of tumor cell growth, we should observe increased expression in SCCHN tumors. Furthermore, if PKC{alpha} up-regulates cyclin E expression, then a corresponding increase in cyclin E protein expression would be observed in SCCHN. We examined PKC{alpha} and cyclin E expression in normal human oral mucosa, dysplastic oral mucosa, and head and neck tumor biopsies by immunohistochemistry. Phosphorylation of the PKC{alpha} at the turn motif site maintains catalytic competence and is required for kinase activity (26, 27); therefore, to assess expression of enzymatically competent PKC{alpha}, samples were also immunostained with anti–phosphorylated antibody directed against Thr638. Analysis of the relative distribution of staining intensity reveals that both PKC{alpha} and phosphorylated PKC{alpha} expression increased progressively from normal to dysplastic to malignant tissue (Fig. 5A and B ; P < 0.0001 by Cuzick's trend test). Both normal and dysplastic tissue show predominant nuclear staining for both anti-PKC{alpha} and anti–phosphorylated PKC{alpha}. In contrast, analysis of the malignant tissue reveals prominent cytoplasmic staining that is not present in the neighboring stroma. These results indicate that PKC{alpha} expression correlates with progression to SCCHN.


Figure 5
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Tissue expression of total and phosphorylated PKC{alpha} and association of PKC{alpha} gene expression with miR-15a expression and tumor recurrence. A, left, proportion of normal, dysplastic, or malignant specimens classified by staining intensity with anti-PKC{alpha} antibody. Representative sections of normal (N), dysplastic (D), and malignant (M) tissue sections stained with anti-PKC{alpha} antibody. Right, proportion of normal, dysplastic, or malignant specimens classified by staining intensity with anti–phosphorylated PKC{alpha} antibody. Representative sections of normal (N), dysplastic (D), and malignant (M) tissue sections stained with anti–phosphorylated PKC{alpha} antibody. Staining intensity is expressed on a four-point scale from 0 to 3+ (see Materials and Methods). B, Kaplan-Meier curve of DFS probability as a function of high (red) versus low (black) PKC{alpha} gene expression by Affymetrix array. Statistical significance was determined by the log-rank test. C, PKC{alpha} transcript and miR-15a levels inversely correlate in individual SCCHN tumor samples (R2 = 0.22). mRNA and miRNA were isolated from 19 SCCHN tumor samples and analyzed for expression of PKC{alpha} transcripts and miR-15a by RT-PCR, as described in Materials and Methods. The results were plotted and analyzed by linear regression analysis for an inverse correlation. The P value reveals statistical significance.

 
The phosphorylated PKC{alpha} antibody also detects phosphorylated PKCβII at a complementary site (Thr641). Therefore, we assessed expression of PKCβII in the same tissue microarray and observed no staining in normal and dysplastic tissue. The SCCHN samples expressed low levels of PKCβII (17% were 1+, 3% were 2+, 0 were 3+; data not shown), suggesting that expression detected using the phosphorylated antibody stems mostly from PKC{alpha}. Moreover, samples staining for PKC{alpha} were highly correlated with the phosphorylated antibody (Spearman's Rho correlation, 0.48; probability that PKC{alpha} and phosphorylated PKC{alpha} are independent, <0.00001).

Cyclin E staining displayed a similar pattern as PKC{alpha}. No expression in normal and dysplastic tissue was detected in tissue microarrays stained with anti–cyclin E antibody, whereas 18% of SCCHN samples were stained positively (10% were 1+, 6% were 2+, 2% were 3+; P < 0.0001; Supplementary Fig. A4A). The relatively low staining intensity in all tissues likely reflects the transient expression of cyclin E in proliferating cells, the heterogeneity in aggressiveness of the malignant tissues examined, and the limited sensitivity of the antibody used. Nevertheless, the increased expression of PKC{alpha} and cyclin E in SCCHN tumor tissue mirrors the PKC{alpha} induction of these proteins in the radioresistant, highly proliferative SCCHN cell line SQ20B.

To assess whether PKC{alpha} expression had prognostic significance, disease-free survival (DFS) analysis was performed in a previously described cohort of 44 patients treated for SCCHN using expression measurements for a PKC{alpha} probe on Affymetrix microarray (15). Median follow-up time was 27 months (range, 3–111 months), and events were recorded as either disease recurrence or death. Because PKC{alpha} expression is measured as a continuous variable, we examined the data using a Cox proportional hazards model using PKC{alpha} expression as a predictor and DFS as outcome measure. In this model, lower PKC{alpha} expression was a significant predictor of longer DFS (P = 0.027). For visualization of data, patient samples were divided based on PKC{alpha} expression (threshold, 0.15) and Kaplan-Meier curves were plotted using DFS as the outcome measure (Fig. 5B). Log-rank analysis reveals that high PKC{alpha} expression was associated with a significantly higher probability of disease recurrence or death (P = 0.0062). In addition, overall survival was also significantly shortened in the high PKC{alpha} expression group (P = 0.0028 by log-rank test or P = 0.017 by Cox proportional hazards model; data not shown). This evidence further substantiates the regulation seen in vitro and suggests that PKC{alpha} mediates SCCHN tumor progression.

Because PKC{alpha} negatively regulates mir-15a in vitro, we examined expression of miR-15a in 29 different SCCHN primary patient tumor samples. Ten of the 29 samples were selected based on availability of tissue for miRNA isolation and relative PKC{alpha} gene expression (Supplementary Fig. S4B) from a previously published data set (15). An inverse relationship was noted between miR-15a expression and PKC{alpha} transcript levels in tumor samples (P = 0.09; Supplementary Fig. S4C). To determine whether this relationship exists within individual SCCHN tumors, an additional 19 samples were analyzed for both PKC{alpha} and miR-15a expression by qRT-PCR (Fig. 5C). The results confirm that PKC{alpha} and miR-15a expressions are inversely related (slope –1.75, P = 0.04). This evidence further substantiates the regulation seen in vitro and suggests that PKC{alpha} mediates its oncogenic effects, at least in part, through miR-15a. In addition, because PKC{alpha} is prognostic for progression in primary SCCHN tumors, these results suggest that miR-15a expression should correlate with DFS.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
These results implicate PKC{alpha} as a key mediator of SCCHN proliferation through activation of MAPK and negative regulation of miR-15a, an inhibitor of cyclin E expression, leading to increased synthesis of cell cycle proteins and enhanced DNA synthesis. Our findings fit a coherent type 4 feed-forward loop (FFL) network (ref. 28; Fig. 6 ). The FFL is initiated by PKC{alpha} with one arm comprising a series of activating reactions and the other arm consisting, in part, of two successive inhibitory reactions that together promote cyclin E expression and DNA synthesis. In the present case, PKC{alpha} activates a driver, MAPK, as well as releasing a brake, miR-15a (Fig. 6B). The key characteristics of FFLs are a delay in the activation of the system upon stimulation and a rapid shut-off upon loss of the stimulus. Both of these features represent key elements that ensure the integrity of DNA synthesis by enabling initiation only when two input conditions are satisfied and causing rapid cessation when either input is lost. However, in cancer cells, the stimulus is constitutively activated so that there is no initial delay in the activation of the cell cycle. It is likely that this type of coherent type 4 FFL network, consisting of a stimulus that activates a positive signal and removes a negative brake, is a general characteristics of cell cycle progression in both normal and tumor cells.


Figure 6
View larger version (9K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Schema illustrating a FFL initiated by PKC{alpha} that regulates DNA synthesis. A, illustration of a typical FFL with X as the initiation signal that stimulates Y activation rapidly and Z activation slowly. Y also stimulates Z resulting in maximal activation of the network. B, the FFL involves PKC{alpha} that activates MAPK, as well as cyclin E translation via inhibition of mir-15a. Note that the "slow" arm of the loop involves negative regulation of a suppressor resulting in forward flow. C, specific components of the FFL. Normal G1-S phase transition in response to mitogenic extracellular stimuli, such as EGF, induces activation of the Raf/MEK/MAPK cascade. We have previously shown that PKC{zeta} mediates EGF activation of MAPK (2). Here, we show that PKC{alpha} can also activate MAPK. This signaling cascade leads to expression of cyclin D complexed with cdk4/cdk6 followed by induction of cyclin E complexed with cdk2; each cyclin-activated kinase in turn phosphorylates Rb. In concert with this, PKC{alpha} activity induced by oncogenic stimuli deregulates cyclin E expression by suppressing miR-15a, an inhibitor of cyclin E. The net effect of PKC{alpha} activation is to trigger a positive feedback loop resulting in up-regulated cyclin E expression and DNA synthesis.

 
Because both PKC{alpha} and cyclin E have been implicated in diverse cancers, these findings could have broader relevance. The mechanism described for up-regulating cyclin E involves inhibition of miR-15a by activated PKC{alpha}. miR-15a and miR-16-1 are located in the 13q14 locus, deleted in the majority of chronic lymphocytic leukemias, consistent with tumor suppressor function (29). Further evidence showed that miR-15a negatively regulates Bcl-2 and, thus, induces apoptosis in a leukemic cell line (30). In pituitary adenomas, suppression of miR-15a and miR16-1 has been associated with tumor growth (31). However, in endocrine pancreatic tumors, the same miRs seem to be overexpressed underscoring the likelihood that their role is tissue-specific (32). A functional screen of the miR16 family that includes miR-15a in HCT116, HeLa, and TOV21G cell lines showed that these miRs negatively regulate cell cycle progression (11). The miR16 family in these cells functioned primarily in feedback regulation, and its inhibition of DNA synthesis was mediated by its cumulative effect on multiple cell cycle targets. In contrast, we show here that, in SCCHN, miR-15a regulation of cyclin E expression is sufficient to account for the negative regulation of DNA synthesis by PKC{alpha}. Our results suggest that miR-15a is a positive prognostic marker based on its relationship with PKC{alpha} expression and may function as a tumor suppressor that negatively regulates cell proliferation.

It is noteworthy that inhibition of PKC{alpha} with siRNA did not mimic all the effects of Gö6976 in vitro, suggesting that other PKCs, such as PKC{varepsilon}, could also play a role in proliferation. Thus, the in vivo effects observed in response to Gö6976 could result in part from inhibition of other PKC isoforms in tumor cells, mouse stromal tissue, or immune cells. PKCβ has been implicated as a mediator of angiogenesis through inhibition of GSK3β (33), but a recent study using Gö6976 and other PKC inhibitors concluded that PKC{alpha}, rather than PKCβ, promotes angiogenesis (34). PKCs also regulate immune cell function (35–37); thus, the contribution of specific isoforms with respect to tumor microenvironment and immune system needs to be further elucidated.

PKC has been shown to regulate cyclins or E2Fs in other cell types, but the outcomes can differ depending upon the specific system. For example, overexpression or 12-O-tetradecanoylphorbol-13-acetate activation of PKC{alpha} in the late G1 phase was shown to inhibit rather than activate E2F activity and DNA synthesis in rat 3Y1 fibroblasts (38). Conversely, constitutively activated PKC{alpha} enhances both cyclins D and E promoter activity in NIH3T3 cells (39). In human keratinocytes, activation of PKC{alpha} has been implicated in cell cycle arrest or terminal differentiation (40–42), whereas in some cancers PKC{alpha} expression is decreased or possesses tumor suppressor function (3). This is not necessarily contradictory to our findings because similar mechanisms may be used in other tissues, but other pathways may also be present that counteract the effect of PKC{alpha} or function as feedback regulators. In SQ20B cells, cyclin E gene expression and proliferation was significantly altered by PKC{alpha}. It is also important to note that the cell lines we characterized were derived from rapidly proliferating, radiation-resistant, highly EGF receptor (EGFR)–expressing tumors, and the signaling cascade elucidated may not be characteristic of less aggressive SCCHN cancers.

Previous work from our laboratory showed that EGF-stimulated PKC{zeta} regulates SCCHN DNA synthesis by contributing to Raf-1/MAPK activation (2). Numerous studies have shown that other PKC isoforms, such as PKC{alpha}, also stimulate Raf-1 activation. Here, we show a complementary mechanism for the oncogenic effects of PKC{alpha}, namely, stimulation of MAPK together with inhibition of miR-15a, leading to up-regulation of cyclin E synthesis. However, unlike PKC{zeta}, PKC{alpha} is activated independently of EGFR (data not shown). The mechanism leading to constitutive PKC{alpha} activation and overexpression in tumors remains to be determined.

Our results highlighted cyclin E as the key target of PKC{alpha} regulation. Cyclin E was able to substantially rescue the inhibition of DNA synthesis upon PKC{alpha} kinase inactivation in our cells. In addition to its role in complex with cdks, cyclin E was recently found to be required for loading the MCM replicative helicase onto replicative origins (replicative licensing) and for transformation by Ras in a manner that is independent of cdks (43). Thus, cyclin E performs dual functions that are both cdk-dependent and cdk-independent, and loss of cyclin E should rapidly prevent DNA synthesis.

Cyclin E overexpression or deregulation has been associated with a number of highly aggressive tumors with poor prognosis, but the mechanisms that have been described differ from the one elucidated here (reviewed in ref. 44). In laryngeal squamous cell carcinomas (LSCC), cyclin E overexpression alone was a prognostic marker for early-stage LSCC and tumors with a combination of high cyclin E and PCNA expression yielded the poorest prognoses (45, 46). Cyclin E overexpression in tumors has been attributed to a number of mechanisms, including loss or mutation of ubiquitin ligases, decreasing the rate of degradation, cyclin E gene amplification, and mutation of oncogenes upstream of the cyclin D-Rb-E2F pathway that normally regulate cell cycle progression (44). Our results contribute an additional mechanism for cyclin E overexpression involving regulation of synthesis by PKC{alpha} via mir-15a.

This study is the first demonstration that PKC{alpha} is a mediator of SCCHN proliferation and a marker of progression and prognosis. Our results indicate that PKC{alpha} is a primary driver of the cancer phenotype that promotes SCCHN development and, thus, represents a novel therapeutic target. These results carry important clinical implications by providing a rationale for the advancement of PKC inhibitors that are currently being developed or investigated through clinical trials. Taken together, this evidence suggests that PKC{alpha} inhibition will yield efficacy in a variety of cancers.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: NIH grants RO1-CA109278-01 (M.R. Rosner), P50 DE11921-0551 (E.E.W. Cohen), DE12322 (M.W. Lingen), DE00470 (M.W. Lingen), and RO1-DE017982-01 (C.H. Chung); American Society of Clinical Oncology (E.E.W. Cohen); Francis L. Lederer Foundation (E.E.W. Cohen); Damon-Runyon Cancer Research Foundation grant CI-28-05 (C.H. Chung); and Cornelius Crane Trust for Eczema Research (M.R. Rosner).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The authors thank Philippe Cluzel and Uri Alon for stimulating discussions, and Xinmin Li, Michael Kubal, Carolyn Pierce, Jing Liu, John Mote, and Shawn Levy for technical assistance.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

E.E.W. Cohen and H. Zhu contributed equally to this work.

8 http://pictar.bio.nyu.edu/ Back

Received 1/30/08. Revised 10/13/08. Accepted 10/28/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Dempsey EC, Newton AC, Mochly-Rosen D, and colleagues Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 2000;279:L429–38.[Abstract/Free Full Text]
  2. Cohen EE, Lingen MW, Zhu B, et al. Protein kinase C {zeta} mediates epidermal growth factor-induced growth of head and neck tumor cells by regulating mitogen-activated protein kinase. Cancer Res 2006;66:6296–303.[Abstract/Free Full Text]
  3. Griner EM, Kazanietz MG. Protein kinase C and other diacylglycerol effectors in cancer. Nat Rev Cancer 2007;7:281–94.[CrossRef][Medline]
  4. Wu TT, Hsieh YH, Wu CC, Hsieh YS, Huang CY, Liu JY. Overexpression of protein kinase C {alpha} mRNA in human hepatocellular carcinoma: a potential marker of disease prognosis. Clin Chim Acta 2007;382:54–8.[CrossRef][Medline]
  5. Dlugosz AA, Cheng C, Williams EK, Dharia AG, Denning MF, Yuspa SH. Alterations in murine keratinocyte differentiation induced by activated rasHa genes are mediated by protein kinase C-{alpha}. Cancer Res 1994;54:6413–20.[Abstract/Free Full Text]
  6. Chmura SJ, Dolan ME, Cha A, Mauceri HJ, Kufe DW, Weichselbaum RR. In vitro and in vivo activity of protein kinase C inhibitor chelerythrine chloride induces tumor cell toxicity and growth delay in vivo. Clin Cancer Res 2000;6:737–42.[Abstract/Free Full Text]
  7. Sclafani RA, Holzen TM. Cell Cycle Regulation of DNA Replication. Annu Rev Genet 2007;41:237–80.[CrossRef][Medline]
  8. Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol 2007;17:118–26.[Medline]
  9. Wu W, Sun M, Zou GM, Chen J. MicroRNA and cancer: current status and prospective. Int J Cancer 2007;120:953–60.[CrossRef][Medline]
  10. He L, He X, Lim LP, et al. A microRNA component of the p53 tumour suppressor network. Nature 2007;447:1130–4.[CrossRef][Medline]
  11. Linsley PS, Schelter J, Burchard J, et al. Transcripts targeted by the microRNA-16 family cooperatively regulate cell cycle progression. Mol Cell Biol 2007;27:2240–52.[Abstract/Free Full Text]
  12. O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 2005;435:839–43.[CrossRef][Medline]
  13. Sylvestre Y, De Guire V, Querido E, et al. An E2F/miR-20a autoregulatory feedback loop. J Biol Chem 2007;282:2135–43.[Abstract/Free Full Text]
  14. Woods K, Thomson JM, Hammond SM. Direct regulation of an oncogenic micro-RNA cluster by E2F transcription factors. J Biol Chem 2007;282:2130–4.[Abstract/Free Full Text]
  15. Slebos RJ, Yi Y, Ely K, et al. Gene expression differences associated with human papillomavirus status in head and neck squamous cell carcinoma. Clin Cancer Res 2006;12:701–9.[Abstract/Free Full Text]
  16. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 2001;98:5116–21.[Abstract/Free Full Text]
  17. Chung CH, Parker JS, Ely K, et al. Gene expression profiles identify epithelial-to-mesenchymal transition and activation of nuclear factor-{kappa}B signaling as characteristics of a high-risk head and neck squamous cell carcinoma. Cancer Res 2006;66:8210–8.[Abstract/Free Full Text]
  18. Shapiro BA, Ray S, Jung E, Allred WT, Bollag WB. Putative conventional protein kinase C inhibitor Godecke 6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)py rrolo(3,4-c)-carbazole] stimulates transglutaminase activity in primary mouse epidermal keratinocytes. J Pharmacol Exp Ther 2002;302:352–8.[Abstract/Free Full Text]
  19. Cai H, Smola U, Wixler V, et al. Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase. Mol Cell Biol 1997;17:732–41.[Abstract/Free Full Text]
  20. Berra E, Diaz-Meco MT, Lozano J, et al. Evidence for a role of MEK and MAPK during signal transduction by protein kinase C{zeta}. EMBO J 1995;14:6157–63.[Medline]
  21. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 2005;102:15545–50.[Abstract/Free Full Text]
  22. Li YY, Wang L, Lu CD. An E2F site in the 5'-promoter region contributes to serum-dependent up-regulation of the human proliferating cell nuclear antigen gene. FEBS Lett 2003;544:112–8.[CrossRef][Medline]
  23. Noya F, Chien WM, Wu X, et al. The promoter of the human proliferating cell nuclear antigen gene is not sufficient for cell cycle-dependent regulation in organotypic cultures of keratinocytes. J Biol Chem 2002;277:17271–80.[Abstract/Free Full Text]
  24. Ohtani K, Iwanaga R, Nakamura M, et al. Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F. Oncogene 1999;18:2299–309.[CrossRef][Medline]
  25. Moldovan GL, Pfander B, Jentsch S. PCNA, the maestro of the replication fork. Cell 2007;129:665–79.[CrossRef][Medline]
  26. Garcia-Paramio P, Cabrerizo Y, Bornancin F, Parker PJ. The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation. Biochem J 1998;333:631–6.[Medline]
  27. Hirai T, Chida K. Protein kinase C{zeta} (PKC{zeta}): activation mechanisms and cellular functions. J Biochem Tokyo 2003;133:1–7.[Abstract/Free Full Text]
  28. Alon U. Network motifs: theory and experimental approaches. Nat Rev Genet 2007;8:450–61.[CrossRef][Medline]
  29. Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of microRNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 2002;99:15524–9.[Abstract/Free Full Text]
  30. Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A 2005;102:13944–9.[Abstract/Free Full Text]
  31. Bottoni A, Piccin D, Tagliati F, Luchin A, Zatelli MC, degli Uberti EC. miR-15a and miR-16-1 down-regulation in pituitary adenomas. J Cell Physiol 2005;204:280–5.[CrossRef][Medline]
  32. Roldo C, Missiaglia E, Hagan JP, et al. MicroRNA expression abnormalities in pancreatic endocrine and acinar tumors are associated with distinctive pathologic features and clinical behavior. J Clin Oncol 2006;24:4677–84.[Abstract/Free Full Text]
  33. Goekjian PG, Jirousek MR. Protein kinase C inhibitors as novel anticancer drugs. Expert Opin Investig Drugs 2001;10:2117–40.[CrossRef][Medline]
  34. Bokhari SM, Zhou L, Karasek MA, Paturi SG, Chaudhuri V. Regulation of skin microvasculature angiogenesis, cell migration, and permeability by a specific inhibitor of PKC{alpha}. J Invest Dermatol 2006;126:460–7.[CrossRef][Medline]
  35. Cataisson C, Pearson AJ, Torgerson S, Nedospasov SA, Yuspa SH. Protein kinase C{alpha}-mediated chemotaxis of neutrophils requires NF-{kappa}B activity but is independent of TNF {alpha} signaling in mouse skin in vivo. J Immunol 2005;174:1686–92.[Abstract/Free Full Text]
  36. Suh KS, Tatunchak TT, Crutchley JM, Edwards LE, Marin KG, Yuspa SH. Genomic structure and promoter analysis of PKC-{delta}. Genomics 2003;82:57–67.[CrossRef][Medline]
  37. Sun Z, Arendt CW, Ellmeier W, et al. PKC-{theta} is required for TCR-induced NF-{kappa}B activation in mature but not immature T lymphocytes. Nature 2000;404:402–7.[CrossRef][Medline]
  38. Nakaigawa N, Hirai S, Mizuno K, Shuin T, Hosaka M, Ohno S. Differential effects of overexpression of PKC {alpha} and PKC {delta}/epsilon on cellular E2F activity in late G1 phase. Biochem Biophys Res Commun 1996;222:95–100.[CrossRef][Medline]
  39. Soh JW, Weinstein IB. Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes. J Biol Chem 2003;278:34709–16.[Abstract/Free Full Text]
  40. Lee YS, Dlugosz AA, McKay R, Dean NM, Yuspa SH. Definition by specific antisense oligonucleotides of a role for protein kinase C{alpha} in expression of differentiation markers in normal and neoplastic mouse epidermal keratinocytes. Mol Carcinog 1997;18:44–53.[CrossRef][Medline]
  41. Tibudan SS, Wang Y, Denning MF. Activation of protein kinase C triggers irreversible cell cycle withdrawal in human keratinocytes. J Invest Dermatol 2002;119:1282–9.[CrossRef][Medline]
  42. Yang LC, Ng DC, Bikle DD. Role of protein kinase C {alpha} in calcium induced keratinocyte differentiation: defective regulation in squamous cell carcinoma. J Cell Physiol 2003;195:249–59.[CrossRef][Medline]
  43. Geng Y, Lee YM, Welcker M, et al. Kinase-independent function of cyclin E. Mol Cell 2007;25:127–39.[CrossRef][Medline]
  44. Hwang HC, Clurman BE. Cyclin E in normal and neoplastic cell cycles. Oncogene 2005;24:2776–86.[CrossRef][Medline]
  45. Anton RC, Coffey DM, Gondo MM, Stephenson MA, Brown RW, Cagle PT. The expression of cyclins D1 and E in predicting short-term survival in squamous cell carcinoma of the lung. Mod Pathol 2000;13:1167–72.[CrossRef][Medline]
  46. Dong Y, Sui L, Tai Y, Sugimoto K, Hirao T, Tokuda M. Prognostic significance of cyclin E overexpression in laryngeal squamous cell carcinomas. Clin Cancer Res 2000;6:4253–8.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
J. Huang, S. Wu, C.-L. Wu, and B. D. Manning
Signaling Events Downstream of Mammalian Target of Rapamycin Complex 2 Are Attenuated in Cells and Tumors Deficient for the Tuberous Sclerosis Complex Tumor Suppressors
Cancer Res., August 1, 2009; 69(15): 6107 - 6114.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cohen, E. E.W.
Right arrow Articles by Rosner, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cohen, E. E.W.
Right arrow Articles by Rosner, M. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online