PKCα is growth inhibitory in non-transformed IECs and has been implicated in tumor suppression in APC/β-catenin mutant intestinal neoplasms [3
]. This study extends previous findings by providing the first demonstration that the tumor suppressive effects of PKCα signaling, as well as early loss of PKCα protein, occur independently of specific genetic alterations or degree of differentiation. The study further demonstrates that an important target of PKCα regulation in CRC cells is the potent mitogen cyclin D1. PKCα is shown to suppress cyclin D1 accumulation in CRC cells independently of β-catenin signaling, and cyclin D1 deficiency is implicated in PKCα-induced inhibition of anchorage-independent growth. Two distinct mechanisms of PKCα-mediated cyclin D1 control are identified: transcriptional repression and blockade of cap-dependent translation. Finally, to our knowledge, this study is the first to report a comprehensive analysis of PKC isozyme expression in a panel of CRC cells, and to validate these cells as models for studies on the PKC enzyme system in intestinal tumors.
PKCα is downregulated in murine intestinal tumors and in human CRC cells that are APC mutant (e.g., ApcMin/+, Apc1638 and KRas/Apc+/1638N mice, and FET/DNR, DLD-1, GEO, HCT-15, and RCA cells) or β-catenin-mutant (e.g., azoxymethane-treated mice and HCT116 and LS180 cells), as well as in neoplastic intestinal tissues and cells that are APC/β-catenin wild-type (i.e., pVillin-KRasV12G and Muc2-/- mice, and RKO cells). PKCα signaling appears to be limiting even in CRC cells that retain expression of the enzyme (e.g., FET, HCT116 and LS180), as indicated by their resistance to the effects of PKC agonists on cyclin D1 expression, a deficiency that can be overcome by exogenous expression of the enzyme. Although the underlying mechanisms remain to be delineated, defective PKCα signaling appears to be a general characteristic of early intestinal tumorigenesis, and alterations in this pathway are likely related to its negative growth effects rather than a secondary consequence of specific alterations in individual tumors.
Previous studies in CaCo-2 [10
] and HT-29 [34
] cells, which are capable of enterocytic differentiation in vitro
and form moderately well- or well-differentiated tumors in mice [36
], demonstrated the ability of PKCα signaling to inhibit anchorage-independent growth. The finding that PKCα expression inhibits colony formation in soft agarose of highly aggressive, poorly differentiated cells (DLD-1, HCT116, and FET/DNR), intermediately aggressive, moderately differentiated cells (RCA), as well as non-aggressive, well-differentiated cells (GEO), argues that PKCα retains its tumor suppressive effects even in advanced tumors. This notion is consistent with evidence that PKCα deficiency not only increases the number of early lesions (i.e., aberrant crypt foci and adenomas) in the murine intestine, but also promotes the development of aggressive intestinal adenocarcinomas in ApcMin/+
Loss of PKCα and upregulation of cyclin D1 are both early events in intestinal tumorigenesis ( and [6
]). Evidence in support of a direct relationship between these events includes (a) reciprocal changes in the abundance of PKCα and cyclin D1 in diverse mouse models of intestinal neoplasia and human CRC cell lines; (b) rapid downregulation of cyclin D1 protein in non-transformed IECs in response to PKCα activation [12
]; (c) the failure of PKC agonists to downregulate cyclin D1 in colon tumor cells, which have limiting levels of PKCα signaling; (d) the ability of PKCα re-expression to reduce steady-state levels of cyclin D1 in CRC cells and to re-establish PKC agonist-mediated downregulation of the protein; and (e) the involvement of cyclin D1 deficiency in PKCα-induced suppression of CRC cell growth in soft agar.
Analysis of the effects of PKCα re-expression in CRC cells has identified two distinct mechanisms of PKCα-induced cyclin D1 repression. In addition to agonist-induced translational effects mediated by activation of 4E-BP1, PKCα re-expression inhibits steady state levels of cyclin D1 by a novel transcriptional mechanism not previously identified in non-transformed IECs. Notably, both of these mechanisms appear to be defective in tumor cells, even in those that retain appreciable levels of PKCα. The fact that the defect could be rescued by expression of exogenous PKCα likely reflects reduced sensitivity of downstream signaling components to levels of PKCα activation or defects in activation of the endogenous enzyme. In regard to the latter possibility, it is interesting to note that the migration of PKCα in SDS-PAGE gels often differed between CRC cells and non-transformed IECs (), pointing to a potential difference in post-translational modification. The precise defects in CRC cells are currently under investigation. Our data also indicate that the transcriptional and translational effects on cyclin D1 can occur independently: while endogenous levels of PKCα agonist activity in CRC cells were sufficient to elicit transcriptional effects following PKCα re-expression, further activation with pharmacological agonists was required for the translational effects. Whether this reflects differential sensitivity to levels of PKC activation or unique effects of different stimuli remains to be determined.
The ability of PKCα to downregulate cyclin D1 in CRC cell lines did not require activated β-catenin signaling and was independent of known genetic changes. While our studies in non-transformed IECs point to a role for PP2A in the translational effects of PKCα [33
], downstream effectors that mediate the reduced steady-state levels of cyclin D1 mRNA are not immediately apparent. A potential target is the epidermal growth factor receptor (EGFR) which can promote cyclin D1 expression through various signaling intermediates [38
]. Inhibition of EGFR activation by PKCα has been noted in a number of systems (e.g., [39
]) and was also seen following re-expression of the isozyme in CRC cells (Supplementary Figure 4A
). However, while this receptor is likely to play a role in the regulation of cyclin D1 under some circumstances, our data point to the involvement of additional factors. PKCα repressed cyclin D1 (both at the steady state level and in response to PKC agonist treatment) in SW620 cells which lack EGFR expression [42
]. Indeed, EGFR-independent mechanisms appear to play a significant role even in cells that express the receptor since (a) PKCα is downregulated in tumors and CRC cell lines with activating mutations in proteins downstream of EGFR (e.g., KRas mutant mice and KRas/PI3K mutant DLD1, HCT116 and HCT-15 cells [44
]), mutations that correlate with insensitivity to EGFR inhibition [44
], and (b) effects of PKCα on cyclin D1 mRNA levels did not correlate with sensitivity of CRC cells to EGFR inhibitors [44
] (e.g., the effect seen in resistant HCT116 and HCT-15 cells was comparable with that in sensitive GEO cells ( and data not shown). These EGFR-independent mechanisms are currently under investigation.
A role for downregulation of cyclin D1 in the tumor suppressive effects of PKCα is consistent with known functions of this cyclin in tumorigenesis [13
]. Overexpression of cyclin D1 mRNA and protein is a common characteristic of various cancers, including those of the intestine, where it appears to be caused by trans
-acting influences as opposed to genetic mutation/rearrangement [46
]. A direct role in intestinal tumorigenesis is supported by evidence that cyclin D1-deficiency (a) reduces the number and size of tumors in ApcMin/+
], (b) inhibits intestinal tumor progression following conditional loss of APC in vivo
], and (c) reverses the transformed phenotype of SW480 CRC cells [15
]. The finding that PKCα-induced downregulation of cyclin D1 in CRC cells does not affect the cell cycle (Supplementary Figure 2
) suggests that levels of this cyclin are not limiting for growth of tumor cells under ‘optimal’ conditions (as seen in SW480 cells [15
]). The high levels of cyclin D1 in CRC cells may, thus, be required to maintain cell growth in the less favorable environments found in tumors.
Previous studies also support a role for PKCδ in tumor suppression in the intestine [47
]. Expression of this isozyme is also reduced/lost in rodent and human intestinal neoplasms and CRC cell lines (this study and [4
]). Our comparative analysis demonstrates that, although PKCδ variably affects CRC cells of different genetic background, PKCα is generally more potent in blocking soft agar colony formation, suppressing cyclin D1 steady-state levels, re-establishing downregulation of cyclin D1 in response to PKC agonists, and mediating translational inhibition in response to PMA. Thus, these two isozymes are likely to mediate their tumor suppressive effects through different mechanisms.
Taken together, our findings point to cyclin D1 repression as an important component of PKCα tumor suppression in intestinal cells. Since PKCα has been shown to have tumor suppressive properties in other tissues (e.g., epidermis [49
], pituitary and thyroid [51
]), these findings may be relevant to multiple tumor types.