PROTEIN KINASE C βII AND PKCι/λ: COLLABORATING PARTNERS IN COLON CANCER PROMOTION AND PROGRESSION

Abstract

We previously demonstrated that elevated expression of either protein kinase CβII (PKCβII) or PKCι/λ enhances colon carcinogenesis in mice. Here we use novel bi-transgenic mice to determine the relative importance of PKCβII and PKCι/λ in colon carcinogenesis in two complimentary models of colon cancer in vivo. Bi-transgenic mice over-expressing PKCβII and constitutively active PKCι (PKCβII/caPKCι) or kinase-deficient, dominant negative PKCι (PKCβII/kdPKCι) in the colon exhibit a similar increase in colon tumor incidence, tumor size and tumor burden in response to azoxymethane (AOM) when compared to non-transgenic littermates. However, PKCβII/kdPKCι mice develop predominantly benign colonic adenomas whereas PKCβII/caPKCι mice develop malignant carcinomas. In contrast, PKCβ deficient (PKCβ^−/−) mice fail to develop tumors even in the presence of caPKCι. Our previous data indicated that PKCβII drives tumorigenesis and proliferation by activating β-catenin/Apc signaling. Consistent with this conclusion, genetic deletion of PKCβ has no effect on spontaneous tumorigenesis in APC^min/+ mice. In contrast, tissue-specific knock out of PKCλ significantly suppresses intestinal tumor formation in APC^min/+ mice. Our data demonstrate that PKCβII and PKCι/λ serve distinct, non-overlapping functions in colon carcinogenesis. PKCβII is required for AOM-induced tumorigenesis, but is dispensable for tumor formation in Apc^Min/+ mice. PKCι/λ promotes tumor progression in both AOM- and APC^min/+-induced tumorigenesis. Thus PKCβII and PKCι, whose expression is elevated in both rodent and human colon tumors, collaborate to drive colon tumor formation and progression, respectively.

Introduction

Colon cancer is the third leading cause of cancer death in the United States with an estimated 150,000 new cases and 50,000 deaths in 2008 (1). Colon carcinogenesis is a multi-step process involving progressive changes in signaling pathways that regulate colonic epithelial cell proliferation, differentiation and survival. Prominent among these changes are those involving PKC signaling pathways (reviewed in (2)). It is well-documented that colon carcinogenesis in rodents and humans is accompanied by specific changes in expression of PKC isozymes (2–6). We have previously shown that PKCβII expression is induced in preneoplastic colonic aberrant crypt foci (ACF), and in tumors in AOM-treated mice (3). Elevated expression of PKCβII by transgenesis induces colonic epithelial hyperproliferation and increased susceptibility to colon carcinogenesis (4). PKCβII overexpression induces decreased GSK-3β activity and increased β-catenin levels in the colonic epithelium in vivo, biochemical changes that are associated with the hyperproliferative phenotype of these mice (4). PKCβII also induces invasion of intestinal epithelial cells via a Ras/Mek-, PKCι/λ, Rac 1-dependent signaling pathway in vitro (7).

Like PKCβII, PKCι/λ expression is induced during AOM-mediated colon carcinogenesis (3, 5). Elevated PKCι/λ expression is observed in AOM-induced tumors but not in ACF suggesting that PKCι/λ may function later in the carcinogenic process (3). PKCι/λ activity regulates susceptibility to oncogenic K-ras- and AOM-mediated colon carcinogenesis in transgenic mice (5) and expression of a constitutively active PKCι allele in the colonic epithelium leads to enhanced carcinogenesis after AOM treatment (5). Thus, both PKCβII and PKCι/λ play important roles in colon carcinogenesis. However, the functional relationship between PKCβII and PKCι/λ signaling during colon carcinogenesis has not been explored. Since overexpression of either PKCβII or PKCι can enhance colon carcinogenesis, it is possible that these PKC isozymes serve redundant functions the carcinogenic process. Alternatively, these PKC isozymes may collaborate to coordinately enhance carcinogenesis. In the present study, we examined the relative contribution of PKCβII and PKCι/λ in AOM-induced colon carcinogenesis and in mutant APC- (Apc^Min/+) mediated intestinal tumorigenesis using compound transgenic mice. Our results reveal that PKCβII and PKCι/λ serve distinct, non-overlapping roles in colon carcinogenesis, conspiring to drive initiation and progression of colon carcinogenesis, respectively. PKCβII plays a requisite role in the earliest stages of carcinogen-induced colon carcinogenesis driving adenoma formation. However, PKCβII is dispensible for Apc^Min/+-mediated tumorigenesis. In contrast, PKCι/λ plays an important role in progression of AOM-induced colon tumors from adenoma to carcinoma, and is necessary for Apc^Min/+-mediated tumorigenesis.

Materials and Methods

Mouse Breeding and Maintenance

Transgenic PKCβII, caPKCι and kdPKCι mice (which express human PKCβII, a constitutively active human PKCι allele or a kinase-deficient, dominant negative human PKCι allele respectively, in the colonic epithelium were generated, genotyped and characterized for transgene expression previously (4, 5). Mice nullizygous for PKCβ (PKCβ^−/− mice) were generated as described previously (8, 9). Bi-transgenic mice homozygous for both PKCβII and caPKCι transgenes (PKCβII/caPKCι mice), the PKCβII and kdPKCι transgenes (PKCβII/kdPKCι mice), or homozygous for both the caPKCι transgene and PKCβ knockout allele (PKCβ^−/−/caPKCι mice) were generated by breeding. These mice were maintained on a C57BL/6 genetic background to avoid potential strain differences in phenotype. Apc^Min/+ mice and mice expressing Cre recombinase under control of the mouse villin 1 promoter (Vil-cre) (10) were obtained from The Jackson Laboratory. Floxed PKCλ (PKCλ^fl/fl) mice expressing a conditional, Cre recombinase-inactivated PKCι knock out allele were generated as previously described (11). Bitransgenic Apc^Min/+/PKCβ^−/−, Apc^Min/+/PKCλ^fl/fl mice and tri-transgenic Apc^Min/+/PKCλ^fl/fl/Vil-cre mice were generated by breeding. Mice were housed in microisolator cages in a pathogen-free barrier facility and maintained at a constant temperature and humidity on a 12-h light/12-h dark cycle. Mice were provided with a standard irradiated rodent chow and filtered water ad libitum throughout the experiments. It should be noted that the term PKCι refers to the human atypical PKC isoform derived from the PRKCI gene. PKCλ refers to the mouse homolog of human PKCι. The term PKCι/λ is used to refer to either the mouse or human atypical PKCι isozyme.

Genetic Analysis of Transgenic Mice

Genotypic analysis was performed using the protocols and primer sets described at the following web sites.¹ The presence of the loxP site in PKCλ and Cre-mediated recombination at this site were detected by PCR analysis as described previously (11).

Carcinogen Exposure and Tumor Analysis

Transgenic mice were enrolled in a standard azoxymethane (AOM) carcinogen protocol at six weeks of age as described previously (3). Briefly, mice were injected intraperitoneally with 10 mg/kg AOM or an equal volume of saline once a week for four weeks. Mice were harvested 36 weeks after the last AOM injection for analysis. Upon sacrifice, the colons were isolated, flushed with cold saline, slit open longitudinally and fixed flat in 10% buffered formalin. After 4 hours, the colons were washed in cold PBS and stored in 70% ethanol at 4°C prior to analysis. Fixed colons were stained briefly with 0.5% methylene blue and evaluated for the presence of tumors under a dissecting microscope using 20-fold magnification. The location and size of each tumor was recorded. Tumor volume was calculated as (length × width × width × 0.526). Colon tumors were isolated, along with adjacent normal epithelium and processed for histopathological examination.

Characterization of Apc^Min/+-mediated intestinal tumor formation

At approximately six weeks of age, Apc^Min/+ and Apc^Min/+ compound transgenic mice were transferred from the standard rodent chow to high fat breeder chow (Teklad S-2335) and maintained on this diet until they were sacrificed at 120 days of age. At the time of sacrifice, the small intestine was isolated, divided evenly into three sections (ileum, duodenum and jejunum). The colon from cecum to rectum was also isolated. All intestinal sections were flushed with cold PBS, slit open longitudinally, and evaluated for tumor formation. Intestinal sections were then fixed flat in 4% formalin and subjected to immunohistochemistry. After deparaffinization and rehydration, sections were processed for antigen retrieval as described by the manufacturer (DAKO) and treated with 3% hydrogen peroxide in methanol to inhibit endogenous peroxidases. β-catenin expression and subcellular localization was detected using mouse monoclonal anti- β-catenin antibody (1:800; BD Transduction Labs) and detected with the Envision+Dual Link DAB detection system (DakoCytomation).

Statistical Analysis

Fisher Exact test was used to compare tumor incidence. t-Test was used to compare the means of tumor volume and tumor burden between experimental groups.

Results and Discussion

PKCβII promotes AOM-induced adenoma formation in a PKCι-independent fashion

We previously demonstrated that transgenic PKCβII mice, which overexpress PKCβII in the intestinal epithelium, are more susceptible to AOM-induced colon carcinogenesis (4). Consistent with our previous results, transgenic PKCβII mice exhibit a statistically significant increase in tumor incidence (20.5%) when compared to their non-transgenic littermates (5%) (Figure 1A). We have also implicated atypical PKCι/λ in AOM-induced colon carcinogenesis in vivo (5). Since PKCι/λ is an important component of at least some PKCβII-mediated signaling in intestinal epithelial cells in vitro (7) it is possible that PKCι/λ is a downstream effector of PKCβII in colon carcinogenesis. To test this hypothesis, we determined whether PKCι/λ is required for PKCβII to stimulate tumor formation in response to AOM using two bi-transgenic mouse lines. The first bi-transgenic line consisted of mice that over-express PKCβII and a constitutively active PKCι transgene in the intestinal epithelium (PKCβII/caPKCι mice). The second line consisted of mice that express PKCβII and a kinase deficient, dominant negative PKCι transgene in the intestinal epithelium (PKCβII/kdPKCι mice). Exposure of these two mouse lines to AOM induced formation of colon tumors at a similar incidence (23.7% vs. 20%) that is indistinguishable from that observed in PKCβII mice (Figure 1B). Though there was a trend toward a higher tumor incidence in PKCβII/caPKCι mice when compared to PKCβII or PKCβII/caPKCι mice, the difference between these genotypes did not reach statistical significance. Taken together, these data demonstrate that PKCβII plays a prominent role in tumor formation in response to AOM and indicate that PKCβII is dominant over PKCι/λ in the process of tumor formation. To directly assess this point, we generated PKCβ knock out mice (PKCβ^−/− mice) expressing constitutively active PKCι (caPKCι) in the intestinal epithelium (PKCβ^−/−/caPKCι mice). When these mice are treated with AOM, they failed to form tumors (Figure 1B) demonstrating that PKCβ is absolutely required for tumor formation and that PKCι/λ cannot substitute for PKCβII in the process of tumor initiation. Quantitative PCR analysis of PKCβ^−/− mice demonstrated that these mice exhibit a selective loss of PKCβ expression with no changes in expression of PKCα, PKCλ or PKCζ (data not shown).

Figure 1. PKCβII drives formation and growth of AOM-induced tumors.

A Transgenic PKCβII mice over-expressing PKCβII in the colonic epithelium exhibit enhanced tumor incidence after exposure to azoxymethane (AOM). The Fisher Exact test was used to compare tumor incidence between genotypes. Non-transgenic mice (Non-Tg; n=40); transgenic PKCβII mice (PKCβII; n=78). B. Expression of constitutively active PKCι (caPKCι) or kinase deficient PKCι (kdPKCι) in transgenic PKCβII mice has little effect on tumor incidence induced by PKCβII. PKCβ knock out (KOβ) mice exhibit no tumors even when expressing caPKCι. (PKCβII/caPKCι, n=40); (PKCβII/dnPKCι, n=38); (PKCβ^−/−/caPKCι, n=40) C and D. Transgenic PKCβII mice expressing either caPKCι or kdPKCι exhibit increased tumor volume (C) and tumor burden (D) when compared to non-Tg mice.

We previously reported that PKCι plays a critical role in the anchorage-independent growth of human non-small cell lung cancer (NSCLC) cells in vitro and NSCLC tumorigenicity in vivo (13). Therefore, it is possible that PKCι/λ contributes to the growth of PKCβII-initiated colon tumors in AOM-treated mice. To test this hypothesis, we assessed tumor volume and tumor burden in non-transgenic, PKCβII/caPKCι and PKCβII/kdPKCι mice. Both PKCβII/caPKCι and PKCβII/kdPKCι mice exhibited an increase in tumor volume (Figure 1C) and tumor burden (Figure 1D) when compared to non-transgenic littermates. The increase in tumor volume and burden in PKCβII/dnPKCι and PKCβII/caPKCι mice was not significantly different, though we observed a trend toward smaller tumors and lower tumor burden in PKCβII/dnPKCι mice. These data clearly indicate that PKCι/λ plays a relatively minor role in the growth of PKCβII-initiated colon tumors. We previously demonstrated that PKCβII drives colon tumor formation by stimulating proliferation of colonic epithelial cells in vivo (4). Our current data demonstrate that PKCβII is required for AOM-induced colon carcinogenesis, and indicate that PKCβII promotes colon tumor formation by promoting tumor proliferation in a manner that is largely independent of PKCι/λ. PKCι/λ, on the other hand, plays a relatively minor role in tumor initiation and growth in the presence of elevated PKCβII and cannot drive tumor formation in the absence of PKCβ.

PKCι/λ drives the adenoma to carcinoma progression in AOM-induced colon tumors

Our initial studies on PKC isozyme expression during AOM-induced colon carcinogenesis demonstrated that PKCβII expression was induced very early in the carcinogenesis process. Elevated PKCβII levels were observed in both aberrant crypt foci (ACF), early pre-neoplastic lesions in the colon, and in subsequent colon tumors (3). We also demonstrated that PKCι/λ expression is elevated in AOM-induced tumors suggesting that elevated PKCι/λ expression is functionally linked to colon carcinogenesis at later stages in this model (5). Consistent with this finding, transgenic caPKCι mice often develop intramucosal carcinomas rather than adenomas when treated with AOM (5). Therefore, we assessed whether PKCλ plays a similar role in tumor progression in our bi-transgenic mice. Pathologic analysis of tumors from PKCβII/kdPKCι and PKCβII/caPKCι mice treated with AOM revealed that PKCβII/kdPKCι mice developed predominantly benign adenomas (7/8 tumors classified as adenoma), retaining significant tissue polarity and crypt-like structure comparable to those produced in non-transgenic mice (Figure 2A and B). In sharp contrast, the majority of tumors (8/11) in PKCβII/caPKCι mice contained foci exhibiting progression to malignant carcinoma (p=0.015 compared to PKCβII/kdPKCι mice). Colon carcinomas in PKCβII/caPKCι mice were characterized by the presence of disorganized sheets of epithelial tumor cells, disruption of normal crypt-like glandular architecture and loss of baso-lateral to apical cell polarity (Figure 2C). These data are consistent with our previous observation regarding PKCι and colon tumor progression (5).

Figure 2. PKCι/λ drives the adenoma to carcinoma progression in AOM-induced colon tumors.

Representative sections of tumors from non-transgenic (Non-Tg) (A), PKCβII/dnPKCι (B) and PKCβII/caPKCι (C) mice were stained with hematoxylin and eosin. Non-Tg and PKCβII/kdPKCι mice develop primarily adenomas which retain crypt-like architecture and epithelial cell polarity. In contrast, tumors from PKCβII/caPKCι mice are often carcinomas characterized by loss of crypt organization and polarity, and formation of disorganized sheets of transformed tumor cells.

PKCβII is dispensable for intestinal tumorigenesis in Apc^Min/+ mice

PKCβII-induced intestinal epithelial cell proliferation is associated with activation of the APC/β-catenin proliferative signaling pathway and is characterized by increased GSK3β activity and stabilization of β-catenin in the colonic epithelium in vivo (4). Our current data are consistent with the hypothesis that PKCβII promotes AOM-induced tumorigenesis by activating the APC/β-catenin proliferative signaling pathway. Transgenic PKCβII mice exhibit decreased GSK3β activity in the colonic epithelium in vivo (4), consistent with the finding that PKCβII can directly phosphorylate GSK3β at Serine 9, a phosphorylation event that inhibits GSK3β activity (14). GSK3β inhibition leads to stabilization of β-catenin by inhibiting APC-mediated proteosomal degradation, providing a plausible mechanism by which PKCβII can stabilize β-catenin. If β-catenin is a critical effector of PKCβII-mediated colon tumorigenesis, one would predict that the requirement for PKCβII to promote tumorigenesis might be alleviated in mice containing an inactivating APC mutation, which serves to stabilize β-catenin independent of GSK3β. To test this hypothesis, we crossed PKCβ^−/− with Apc^Min/+ mice to produce Apc^Min/+/PKCβ^−/− mice. As expected, Apc^Min/+/PKCβ^−/− mice exhibit undetectable levels of PKCβII mRNA in the intestinal tract as determined by QPCR, confirming the lack of PKCβ expression (Figure 3A). In contrast, Apc^Min/+ mice express abundant PKCβII mRNA in the intestine (Figure 3A). Apc^Min/+ and Apc^Min/+/PKCβ^−/− mice exhibited no change in expression of the other PKC isozymes implicated in colon tumorigenesis, PKCα, PKCι or PKCζ (Figure 3A). Tumor analysis revealed no change in the number of intestinal tumors between these two genotypes (Figure 3B). Further analysis of tumor development in the colon and the small intestine separately also revealed no change in tumor number or distribution in these organs (Figure 3C). Likewise, there was no significant change in tumor number or distribution within the three major regions of the small intestine, the duodenum, jejunum and ileum in these mice (Figure 3D). Analysis of tumor burden and average tumor size also revealed no change between these genotypes (data not shown). We conclude that PKCβ is dispensable for intestinal tumorigenesis in Apc^Min/+ mice. Our data are consistent with the conclusion that PKCβII promotes AOM-mediated colon carcinogenesis by stabilizing β-catenin and driving tumor proliferation, whereas in Apc^Min/+ mice, β-catenin is stabilized by loss of APC function, thereby eliminating the need for PKCβII.

Figure 3. PKCβ is dispensable for tumor formation in Apc^Min/+ mice.

A. Bi-transgenic Apc^Min/+/PKCβ^−/− mice exhibit profound loss of PKCβ expression in the intestine. Intestinal epithelium from Apc^Min/+ and Apc^Min/+/PKCβ^−/−; mice was isolated and analyzed by QPCR for PKCα, PKCβII, PKCλ and PKCζ mRNA abundance. * denotes p=0.0003; n=5. B–D. Apc^Min/+ and Apc^Min/+/PKCβ^−/− mice develop equal numbers of tumors throughout the intestinal tract (B). Tumors from Apc^Min/+ and Apc^Min/+/PKCβ^−/− mice are distributed in the same pattern within the colon and small intestine (C) and within the sub-regions of the small intestine (D). B–D. n=13 for Apc^Min/+; n=20 for Apc^Min/+/PKCβ^−/−.

PKCι/λ is important for intestinal tumorigenesis in Apc^Min/+ mice

We next assessed whether PKCι/λ plays a role in intestinal tumorigenesis in Apc^Min/+ mice. To address this question, we generated a compound transgenic mouse line consisted of Apc^Min/+ mice crossed to mice harboring a floxed PKCλ allele that supports conditional, Cre recombinase-mediated inactivation of the PKCλ allele (Apc^Min/+/PKCλ^fl/fl mice). Apc^Min/+/PKCλ^fl/fl mouse were then crossed to a villin-Cre mouse that expresses Cre-recombinase constitutively in the intestinal tract under the control of the tissue-specific villin promoter to produce Apc^Min/+/PKCλ^fl/fl/villin Cre mice. Apc^Min/+/PKCλ^fl/fl littermates from this cross served as a negative control in these experiments. QPCR analysis of intestinal epithelium isolated from Apc^Min/+/PKCλ^fl/fl mice and Apc^Min/+/PKCλ^fl/fl/villin Cre mice demonstrated a significant decrease in PKCλ mRNA expression in the intestinal epithelium of Apc^Min/+/PKCλ^fl/fl/villin Cre mice when compared to Apc^Min/+/PKCλ^fl/fl mice (Figure 4A). Despite the loss of PKCλ expression in the intestinal tract, Apc^Min/+/PKCλ^fl/fl/villin Cre mice were viable and showed no morbidity or breeding problems. The intestinal epithelium of these mice exhibited no obvious morphological changes in crypt or villus architecture, polarity or organization (Figure 4B). Furthermore, the baso-lateral membrane distribution and intensity of β-catenin in the intestine of these mice was indistinguishable from that observed in Apc^Min/+/PKCλ^fl/fl mice (Figure 4C). Thus, Cre mediated knock out of PKCλ in the intestinal epithelium has no obvious detrimental consequence on intestinal epithelial tissue architecture, function or polarity.

Figure 4. Tissue-specific knock out of PKCλ does not overtly affect intestinal crypt architecture or polarity.

A Tissue-specific, Cre-recombinase-mediated knockout of PKCλ in the intestinal epithelium. QPCR analysis of RNA isolated from the intestinal epithelium of Apc^Min/+/PKCλ^fl/fl and Apc^Min/+/PKCλ^fl/fl/villin-Cre mice demonstrates loss of PKCλ expression as a result of Cre-mediated recombination. * denotes p=0.002. n=3. B and C. Apc^Min/+/PKCλ^fl/fl (B) and Apc^Min/+/PKCλ^fl/fl/villin-Cre (C) mice stained by H&E (left panels) reveals no overt changes in crypt-villus structure. β-catenin staining (right panels) is unchanged in Apc^Min/+/PKCλ^fl/fl/villin-Cre mice when compared to Apc^Min/+/PKCλ^fl/fl mice indicating that cellular polarity is not disrupted by PKCλ knock out.

Apc^Min/+/PKCλ^fl/fl mice develop numerous tumors that carpet the intestinal epithelium (Figure 5A). However, Apc^Min/+/PKCλ^fl/fl/villin Cre mice develop significantly fewer intestinal tumors than Apc^Min/+/PKCλ^fl/fl mice (Figure 5A). Interestingly, Apc^Min/+/PKCλ^fl/fl/villin-Cre mice exhibited a decrease in tumor number in the small intestine but not the colon (Figure 5B). It is unclear whether this observation reflects a differential function for PKCλ in the colon and small intestine, or is due to the smaller number of colon tumors induced in the colon in this model. Apc^Min/+/PKCλ^fl/fl/villin-Cre mice developed fewer tumors in all regions of the small intestine indicating that PKCι/λ is important for tumorigenesis throughout the small intestine (Figure 5C). Pathologic and immunohistochemical analysis of tumors from Apc^Min/+/PKCλ^fl/fl and Apc^Min/+/PKCλ^fl/fl/villin-Cre mice revealed that both genotypes produced adenomas exhibiting elevated expression and nuclear localization of β-catenin characteristic of the Apc^Min/+ phenotype (Figure 5D). Thus, our data demonstrate that PKCι/λ plays an important role in Apc^Min/+ tumorigenesis.

Figure 5. Tissue-specific knock out of PKCλ inhibits tumorigenesis in Apc^Min/+ mice.

A Genetic deletion of PKCλ inhibits intestinal tumorigenesis. Apc^Min/+/PKCλ^fl/fl mice develop numerous tumors throughout the intestinal epithelium whereas Apc^Min/+/PKCλ^fl/fl/villin-Cre mice develop significantly fewer tumors throughout the intestinal tract. * denotes p=0.000002. B. Genetic deletion of PKCλ inhibits tumor formation in the small intestine but not the colon. * denotes statistically significant difference p=0.000004. C. Deletion of PKCλ inhibits tumorigenesis throughout the small intestine. *p=0.001, ** p=0.01. A–C. n=19 for Apc^Min/+/PKCλ^fl/fl mice; n=33 for Apc^Min/+/PKCλ^fl/fl/villin-Cre mice. D. β-catenin staining of intestinal tumors from Apc^Min/+/PKCλ^fl/fl and Apc^Min/+/PKCλ^fl/fl/villin-Cre mice demonstrating elevated expression and nuclear localization in tumor from both genotypes.

Our results are interesting in light of the observed changes in PKC isozyme expression in AOM- and Apc^Min/+-induced carcinogenesis models. We previously demonstrated that both PKCβII and PKCι/λ expression is elevated during AOM-induced colon carcinogenesis, albeit with different kinetics (3). Our current study demonstrates the importance of both of these changes in driving distinct, complimentary aspects of colon tumor development. We have also observed a loss of PKCα expression in AOM-induced ACF and tumors (3), and intestinal tumors from APC^min mice exhibit changes in PKC isozyme expression similar to those observed in AOM-induced colon tumors (6). Specifically, PKCβ and PKCι/λ expression is elevated in intestinal tumors of Apc^Min/+ mice whereas the expression of PKCα and PKCζ is reduced in these tumors when compared to surrounding normal intestinal epithelium (6). Although the functional importance of the loss of PKCα expression has not yet been elucidated in the AOM model of colon carcinogenesis, PKCα loss drives tumorigenesis in the context of Apc^Min/+ (6). Nullizygous PKCα (PKCα^−/−) mice exhibit enhanced Apc^Min/+-mediated tumorigenesis, and spontaneous intestinal tumor formation in the absence of Apc^Min/+, demonstrating that PKCα acts as a tumor suppressor in the intestinal tract (6). In contrast, nullizygous PKCζ mice exhibited no change in susceptibility to Apc^Min/+mediated tumors.

Our present results demonstrate that atypical PKCζ and PKCι/λ play distinct, non-redundant roles in colon carcinogenesis. Whereas PKCζ is dispensable for Apc^Min/+ mediated tumorigenesis (6), PKCι plays a key role in both AOM-and Apc^Min/+-induced carcinogenesis. These results are consistent with our findings in non-small cell lung cancer (NSCLC) where we have shown that PKCι, but not PKCζ, is specifically overexpressed in human primary NSCLC tumors and NSCLC cell lines (13, 15). Expression of dominant negative, kdPKCι or RNAi-mediated knock down of PKCι expression, leads to inhibition of anchorage-independent growth and invasion of NSCLC cell in vitro, and loss of NSCLC cell tumorigenicity in vivo (13, 15, 16). In contrast, RNAi mediated knock down of PKCζ has no effect on either anchorage-independent growth or invasion NSCLC cells (16). Our studies indicate that analysis of PKC isozyme expression patterns in various tumor models provides important clues to the role of specific PKC isozymes in tumorigenesis; however transgenic models such those employed here and in our previous studies (4, 5, 6) are essential to definitively investigate the functional role of individual PKC isozymes, and of changes in PKC isozyme expression, in physiologic and pathologic processes in vivo. Our data also demonstrate for the first time that two PKC isozymes implicated in colon carcinogenesis, PKCβII and PKCι/λ, play distinct, non-redundant roles in colon tumorigenesis. Furthermore, our data provide compelling evidence that individual PKC isozymes often cannot compensate for the loss of another, highly related PKC isozyme to perform a similar function.

Our results have important implications for the use of therapeutic agents targeting the PKCβ and PKCι/λ isozymes since drugs targeting both of these PKC isozymes are currently being evaluated in clinical trials. Enzastaurin is a potent, selective small molecule inhibitor of PKCβ that has shown clinical activity in glioma and B-cell lymphoma (17–19). Enzastaurin has also shown promise in pre-clinical xenograft models of hepatocellular carcinoma and colon carcinoma (20, 21). Our results indicate that Enzastaurin may also be useful in colon cancer, particularly in the setting of chemoprevention. In this regard, we have found that colonic PKCβII is a relevant target of the chemopreventive activity of dietary ω-3 fatty acids (12). Enzastaurin is extremely well tolerated (22, 23), making it a viable candidate for chemoprevention/chemo-intervention in the context of high risk colon cancer patients. However, since APC/β-catenin mutations are a prevalent event in both sporadic and familial colon cancers, PKCβ-directed therapy may be most effective in very early chemoprevention strategies against colon cancer, prior to acquisition of an activating mutation in β-catenin or loss of Apc tumor suppressor activity. In contrast, our data indicate that PKCι may be a more useful chemotherapeutic target for the treatment of advanced colon cancers that have acquired Apc/β-catenin and/or K-ras mutations. We have shown that PKCι is required for oncogenic K-ras mediated transformation both in the colon and the lung in vitro and in vivo (5, 13). Our present data demonstrate that PKCι/λ is also necessary for Apc/β-atenin mediated tumorigenesis in vivo. Since K-ras, β-catenin and Apc mutations are observed in the majority of human colon cancers, PKCι inhibition may be an attractive strategy for treatment of these tumors. We have recently discovered a novel small molecule inhibitor of PKCι/λ, aurothiomalate (ATM), that shows good anti-tumor activity non-small cell lung cancer (NSCLC) in pre-clinical models (24–26). We recently demonstrated that elevated expression of PKCι in NSCLC is associated with enhanced response to PKCι-targeted therapy with ATM (26). Since PKCι expression is elevated in AOM-induced colon tumors (5), tumors in Apc^Min/+ mice (6) and primary human colon tumors (5), these tumors may also be responsive to PKCι-targeted therapy. Future studies will be required to assess the efficacy of Enzastaurin and ATM in colon carcinogenesis in these models.

Abbreviations used

ACF

aberrant crypt foci

AOM

azoxymethane

H&E

hematoxylin and eosin