Small populations of cancer cells within multiple types of solid tumors have been identified based on cell surface marker expression and other phenotypic and functional characteristics. These subpopulations of tumor cells have often demonstrated a >100-fold increase in tumorigenic potential, compared to the remainder of the cells in the tumor. Furthermore, tumors that form from these cancer stem cells are indistinguishable from the human tumors in which they originate, indicating that the tumor-initiating cells are stem cell-like in their ability to self-renew and give rise to a heterogeneous cell population. Much recent data suggests that elimination of these cancer stem cells, which are typically resistant to conventional therapies, represents the most formidable barrier to curing solid tumors [1, 4, 5, 32, 33, 35]. CSCs, or subclones thereof, are the most likely perpetrators of invasion and metastasis [6, 62].
Recent findings have shown the existence of activated and quiescent repertoires of stem cells in established tumor cell lines as well as primary tumor cell isolates, and their ability to interchange between these conditions . Sphere-forming assays (SFA) are believed to evaluate the differentiation and self-renewal capabilities of a tumor cell population by assessing the potential of a tumor cell to behave like a stem cell, and are widely used in stem cell studies . Sphere-forming assays have been commonly used to retrospectively identify normal and cancer stem cells, and measure stem cell/early progenitor activity in multiple types of solid cancers [38, 63, 64]. Increased expression of “stemness-related genes”  was observed when comparing solid tumor cell lines grown as 3D spheroids to monolayers.
Our identification of PKCδ as a critical mediator of survival in multiple types of solid tumors, including prostate, breast, lung, pancreatic, neuroendocrine and melanomas [45–48] raised the possibility that CSC populations might be similarly dependent upon the activity of this enzyme. The effects of PKCδ inhibition on CSCs, however, had not been previously explored.
We first validated PKCδ as a target in diverse CSCs by demonstrating here that specific and selective down-regulation of PKCδ by shRNA was sufficient to prevent the growth of human breast, pancreatic and prostate cancer stem-like cell cultures, and to induce cytotoxicity.
Potential therapeutic translation of this synthetic lethal approach required the development of small molecule probes. As no PKCδ-selective inhibitors had been developed to date, we initially used pharmacophore modeling and docking of rottlerin, a well-established but not highly-specific inhibitor of PKCδ, into the crystal structure of PKCθ, to identify regions of the molecule important for PKCδ-selectivity. The initial new molecule showing activity against PKCδ (KAM1) was formed by combining structural elements of the broad spectrum protein kinase inhibitor staurosporine and rottlerin. The chromene portion of rottlerin was combined with the carbazole portion of staurosporine to produce KAM1 . KAM1 was further modified to develop 36 new analogs, including BJE6-106, which inhibits PKCδ with an IC50 value of 50 nM and is approximately 1000-fold selective versus PKCα. Specificity for PKCδ over “classical” PKC isoforms, like PKCα, is important, as inhibition of PKCα is generally toxic to all cells, normal and malignant, and would render these inhibitors non-“tumor-targeted”. We have shown that B106 exerts potent cytotoxic activity against N-Ras-mutant human melanomas and B-Raf-mutant melanoma lines that have developed resistance to B-Raf inhibitors by aberrant activation of alternative Ras signaling pathways [48, 55].
We demonstrate here that first, second and third generation PKCδ inhibitors (exemplified by rottlerin, KAM1 and BJE6-106, respectively), inhibit the growth of human cancer stem-like cell cultures isolated from tumors, as well as CSC-like cells derived from cell lines by spheroid formation on non-adherent surfaces. Our prior studies would have predicted that the CSC isolates or spheroids derived from cell lines that contained activating mutations of N-Ras or K-Ras would likely be susceptible to PKCδ suppression (e.g., the K-Ras mutant pancreatic carcinomas and the N-Ras mutant melanomas). The reason for the susceptibility of the stem-like tumor cells containing wt-Ras alleles, however, was not immediately apparent. One reason for their susceptibility is likely to be upregulation of Ras effector pathways (MEK-ERK or PI3K/AKT signaling) in CSC spheres derived from cell lines, compared to the non-CSC parental cultures. We have reported previously that isolated activation of the MEK-ERK effector pathway or the PI3K/AKT effector pathway was sufficient to make cells dependent upon PKCδ for survival [45–47]. The finding of higher levels of Ras effector pathway activation in the CSC sphere subpopulation compared to the parental cells may also explain why in at least one instance (MCF7) the sphere-forming CSC cells were substantially more susceptible to PKCδ inhibition than non-CSC cells population. Interestingly, a recent report has identified a requirement for PKCδ in erbB2-driven proliferation of breast cancer cells , and erbB2 drives aberrant Ras pathway signaling. Furthermore, activation of MAPK pathways in basal-like breast cancers has been reported to promote a cancer stem cell-like phenotype , and activation of Ras/MAPK signaling was reported to protect breast cancer stem cells from certain stem-cell targeted drugs . Collectively, these reports, together with our findings, suggest that a PKCδ-targeted approach to breast cancer stem cell populations, which exploits a synthetic lethal interaction with aberrant Ras signaling, may be particularly effective.
Inhibitory effects of PKCδ suppression on the IL6-Stat3 axis, which is critical for CSC genesis or maintenance in a number of tumor cells types [69–71], may also contribute to the actions of PKCδ inhibition on CSC growth and survival, and will be reported separately.
Epithelial-to-mesenchymal transition (EMT), induced either by paracrine signaling from cancer-associated fibroblasts (CAFs) or neighboring tumor cells, has been associated with the acquisition of a stem cell phenotype . In culture, when immortalized normal or transformed human mammary epithelial cells (HMECs) are stimulated to undergo an epithelial-to-mesenchymal transition (EMT), the transition confers stem-like cell properties upon normal or transformed epithelial cells in culture, partly because the cells acquire a CD44+/CD24 (low) phenotype, similar to breast cancer stem cells.
The idea that cancer cells might reversibly transition between epigenetically-defined tumorigenic and non-tumorigenic states is of interest in part because mechanisms that generate reversible heterogeneity can confer resistance to therapies [73, 74]. We took advantage of a previously-established cell line model system for breast cancer EMT, which consists of a parental spontaneously-immortalized mammary epithelial cell line, MCF 10A (M1), and one of its derivatives, MCF 10C (M3), derived from a xenograft in nude mice that progressed to carcinoma [53, 54]. These cell lines were previously reported to exhibit distinct tumorigenic properties when re-implanted in nude mice; MCF 10A is non-tumorigenic, while MCF 10C forms low-grade, well-differentiated carcinomas [53, 54, 60]. Furthermore, MCF 10C has acquired phenotypic changes consistent with mesenchymal morphology and gene and protein expression patterns characteristic of EMT, including expression of mesenchymal markers (fibronectin, vimentin, and N-cadherin) with concomitant downregulation of E-cadherin, β-catenin, and γ-catenin. MCF 10C also expresses high levels of Nanog, and Sox4, which are markers of cancer stem cells . We found that the mesenchymal, CSC-like MCF 10C subline was much more sensitive to PKCδ inhibitors than the epithelial-like “normal” MCF 10A cells from which they were derived. Furthermore, the MCF 10C line acquired the capacity to efficiently form spheroids when grown in non-adherent conditions, and this tumor spheroid formation was inhibited by inhibition of PKCδ activity.