Protein Kinase C βII and PKCι/λ: Collaborating Partners in Colon Cancer Promotion and Progression Free

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 multistep process involving progressive changes in signaling pathways that regulate colonic epithelial cell proliferation, differentiation, and survival. Prominent among these changes are those involving protein kinase C (PKC) signaling pathways (reviewed in ref. 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 azoxymethane (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, Rac1-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. Because overexpression of either PKCβII or PKCι can enhance colon carcinogenesis, it is possible that these PKC isozymes serve redundant functions in 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-mediated (Apc^Min/+) intestinal tumorigenesis using compound transgenic mice. Our results reveal that PKCβII and PKCι/λ serve distinct, nonoverlapping 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.

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). Bitransgenic 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; ref. 10) were obtained from The Jackson Laboratory. Floxed PKCλ (PKCλ^fl/fl) mice expressing a conditional, Cre recombinase–inactivated PKCι knockout allele were generated as previously described (11). Bitransgenic Apc^Min/+/PKCβ^−/−, Apc^Min/+/PKCλ^fl/fl mice, and tritransgenic 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 homologue 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.³

Carcinogen exposure and tumor analysis. Transgenic mice were enrolled in a standard AOM carcinogen protocol at age 6 wk as described previously (3). Briefly, mice were injected i.p. with 10 mg/kg AOM or an equal volume of saline weekly for 4 wk. Mice were harvested 36 wk 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 h, the colons were washed in cold PBS and stored in 70% ethanol at 4°C before 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 histopathologic examination.

Characterization of Apc^Min/+-mediated intestinal tumor formation. At approximately ages 6 wk, 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 ages 120 d. 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. Student's t test was used to compare the means of tumor volume and tumor burden between experimental groups.

PKCβII promotes AOM-induced adenoma formation in a PKCι-independent fashion. We previously shown 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 with their nontransgenic littermates (5%; Fig. 1A). We have also implicated atypical PKCι/λ in AOM-induced colon carcinogenesis in vivo (5). Because 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 bitransgenic mouse lines. The first bitransgenic line consisted of mice that overexpress 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% versus 20%) that is indistinguishable from that observed in PKCβII mice (Fig. 1B). Although there was a trend toward a higher tumor incidence in PKCβII/caPKCι mice when compared with PKCβII or PKCβII/kdPKCι mice, the difference between these genotypes did not reach statistical significance. Taken together, these data show 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 knockout 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 (Fig. 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 showed that these mice exhibit a selective loss of PKCβ expression with no changes in expression of PKCα, PKCλ, or PKCζ (data not shown).

We previously reported that PKCι plays a critical role in the anchorage-independent growth of human NSCLC cells in vitro and NSCLC tumorigenicity in vivo (12). 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 nontransgenic, PKCβII/caPKCι and PKCβII/kdPKCι mice. Both PKCβII/caPKCι and PKCβII/kdPKCι mice exhibited an increase in tumor volume (Fig. 1C) and tumor burden (Fig. 1D) when compared with nontransgenic littermates. The increase in tumor volume and burden in PKCβII/kdPKCι and PKCβII/caPKCι mice was not significantly different, although we observed a trend toward smaller tumors and lower tumor burden in PKCβII/kdPKCι mice. These data clearly indicate that PKCι/λ plays a relatively minor role in the growth of PKCβII-initiated colon tumors. We previously showed that PKCβII drives colon tumor formation by stimulating proliferation of colonic epithelial cells in vivo (4). Our current data show 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 adenoma to carcinoma progression in AOM-induced colon tumors. Our initial studies on PKC isozyme expression during AOM-induced colon carcinogenesis showed that PKCβII expression was induced very early in the carcinogenesis process. Elevated PKCβII levels were observed in both ACF, early preneoplastic lesions in the colon, and in subsequent colon tumors (3). We also showed 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 bitransgenic 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 of 8 tumors classified as adenoma), retaining significant tissue polarity and crypt-like structure comparable with those produced in nontransgenic mice (Fig. 2A and B). In sharp contrast, the majority of tumors (8 of 11) in PKCβII/caPKCι mice contained foci exhibiting progression to malignant carcinoma (P = 0.015 compared with 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 basolateral to apical cell polarity (Fig. 2C). These data are consistent with our previous observation regarding PKCι and colon tumor progression (5).

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 decreased 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 (13). 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 (Fig. 3A). In contrast, Apc^Min/+ mice express abundant PKCβII mRNA in the intestine (Fig. 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 (Fig. 3A). Tumor analysis revealed no change in the number of intestinal tumors between these two genotypes (Fig. 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 (Fig. 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 (Fig. 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.

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 showed a significant decrease in PKCλ mRNA expression in the intestinal epithelium of Apc^Min/+/PKCλ^fl/fl/villin Cre mice when compared with Apc^Min/+/PKCλ^fl/fl mice (Fig. 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 morphologic changes in crypt or villus architecture, polarity, or organization (Fig. 4B). Furthermore, the basolateral 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 (Fig. 4C). Thus, Cre-mediated knockout of PKCλ in the intestinal epithelium has no obvious detrimental consequence on intestinal epithelial tissue architecture, function, or polarity.

Apc^Min/+/PKCλ^fl/fl mice develop numerous tumors that carpet the intestinal epithelium (Fig. 5A). However, Apc^Min/+/PKCλ^fl/fl/villin Cre mice develop significantly fewer intestinal tumors than Apc^Min/+/PKCλ^fl/fl mice (Fig. 5A). Interestingly, Apc^Min/+/PKCλ^fl/fl/villin-Cre mice exhibited a decrease in tumor number in the small intestine but not the colon (Fig. 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 (Fig. 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 (Fig. 5D). Thus, our data show that PKCι/λ plays an important role in Apc^Min/+ tumorigenesis.

Our results are interesting in light of the observed changes in PKC isozyme expression in AOM- and Apc^Min/+-induced carcinogenesis models. We previously showed that both PKCβII and PKCι/λ expression is elevated during AOM-induced colon carcinogenesis, albeit with different kinetics (3). Our current study shows 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 with 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 show that atypical PKCζ and PKCι/λ play distinct, nonredundant 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 NSCLC where we have shown that PKCι, but not PKCζ, is specifically overexpressed in human primary NSCLC tumors and NSCLC cell lines (12, 14). Expression of dominant-negative kdPKCι or RNAi-mediated knockdown 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 (12, 14, 15). In contrast, RNAi mediated knockdown of PKCζ has no effect on either anchorage-independent growth or invasion NSCLC cells (15). 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 used here and in our previous studies (4–6) are essential to definitively investigate the functional role in vivo. Our data also show for the first time that two PKC isozymes implicated in colon carcinogenesis, PKCβII and PKCι/λ, play distinct, nonredundant 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 because 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 (16–18). Enzastaurin has also shown promise in preclinical xenograft models of hepatocellular carcinoma and colon carcinoma (19, 20). 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 (21). 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, because 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, before 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, 12). Our present data show that PKCι/λ is also necessary for Apc/β-catenin mediated tumorigenesis in vivo. Because 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), which shows good antitumor activity NSCLC in preclinical models (24–26). We recently showed that elevated expression of PKCι in NSCLC is associated with enhanced response to PKCι-targeted therapy with ATM (26). Because 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.

No potential conflicts of interest were disclosed.

Grant support: National Cancer Institute CA081436 (A.P. Fields) and CA094122 (N.R. Murray).

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.

We thank Pam Kreinest and Brandy Edenfield for tissue processing and immunohistochemistry, and Shelly Calcagno for assistance with the expression analysis of transgenic mice.