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Protein kinase C-δ inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo.

Chen Z, Forman LW, Williams RM, Faller DV - BMC Cancer (2014)

Bottom Line: Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture.PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours.Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Center, Boston University School of Medicine, K-712C, 72 E, Concord St,, Boston, MA 02118, USA. dfaller@bu.edu.

ABSTRACT

Background: A subpopulation of tumor cells with distinct stem-like properties (cancer stem-like cells, CSCs) may be responsible for tumor initiation, invasive growth, and possibly dissemination to distant organ sites. CSCs exhibit a spectrum of biological, biochemical, and molecular features that are consistent with a stem-like phenotype, including growth as non-adherent spheres (clonogenic potential), ability to form a new tumor in xenograft assays, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. PKCδ is a novel class serine/threonine kinase of the PKC family, and functions in a number of cellular activities including cell proliferation, survival or apoptosis. PKCδ has previously been validated as a synthetic lethal target in cancer cells of multiple types with aberrant activation of Ras signaling, using both genetic (shRNA and dominant-negative PKCδ mutants) and small molecule inhibitors. In contrast, PKCδ is not required for the proliferation or survival of normal cells, suggesting the potential tumor-specificity of a PKCδ-targeted approach.

Methods: shRNA knockdown was used validate PKCδ as a target in primary cancer stem cell lines and stem-like cells derived from human tumor cell lines, including breast, pancreatic, prostate and melanoma tumor cells. Novel and potent small molecule PKCδ inhibitors were employed in assays monitoring apoptosis, proliferation and clonogenic capacity of these cancer stem-like populations. Significant differences among data sets were determined using two-tailed Student's t tests or ANOVA.

Results: We demonstrate that CSC-like populations derived from multiple types of human primary tumors, from human cancer cell lines, and from transformed human cells, require PKCδ activity and are susceptible to agents which deplete PKCδ protein or activity. Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture. PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours. Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.

Conclusions: These findings suggest that the novel PKC isozyme PKCδ may represent a new molecular target for cancer stem cell populations.

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Effects of PKCδ inhibitors on growth and spheroid formation in non-transformed and transformed human breast epithelial cells. MCF 10A cells and cells from the derived tumorigenic line MCF 10C (also called M3), were grown to 80% confluence in 96-well plates and then exposed to rottlerin at concentrations ranging from 1 to 20 μM (A) or to BJE6-106 at concentrations ranging from 0.1 to 20 μM (B). The corresponding equivalent volume of solvent (DMSO) was used as a vehicle control (Vehicle). After 24, 48 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control (vehicle) values were normalized to 100%. Error bars represent SEM. p values for comparison between vehicle and PKCδ inhibitors on MCF 10A cell number only reached significance (p < 0.05) at 48 hr at 20 μM for rottlerin, and at 1 μM for BJE6-106. In contrast, significant effects of the inhibitors on the MCF 10C cells were observed as early as 24 hr for rottlerin (at 5 μM) and for BJE6-106 (at 0.1 μM). (C) MCF 10A and MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, and spheroid formation was assessed at days 10 and 21. Representative photographs are shown. (D) MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, in the presence of rottlerin (5 μM), or BJE6-106 (1 μM or 5 μM), or DMSO vehicle (Control). Tumor spheroids were enumerated at 10 days. Representative photographs are shown. (E) Spheroid numbers were normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%) and plotted. Error bars represent SEM. p values for comparison between vehicle and rottlerin or BJE6-106 effects on spheroid number were significant (p < 0.001).
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Figure 5: Effects of PKCδ inhibitors on growth and spheroid formation in non-transformed and transformed human breast epithelial cells. MCF 10A cells and cells from the derived tumorigenic line MCF 10C (also called M3), were grown to 80% confluence in 96-well plates and then exposed to rottlerin at concentrations ranging from 1 to 20 μM (A) or to BJE6-106 at concentrations ranging from 0.1 to 20 μM (B). The corresponding equivalent volume of solvent (DMSO) was used as a vehicle control (Vehicle). After 24, 48 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control (vehicle) values were normalized to 100%. Error bars represent SEM. p values for comparison between vehicle and PKCδ inhibitors on MCF 10A cell number only reached significance (p < 0.05) at 48 hr at 20 μM for rottlerin, and at 1 μM for BJE6-106. In contrast, significant effects of the inhibitors on the MCF 10C cells were observed as early as 24 hr for rottlerin (at 5 μM) and for BJE6-106 (at 0.1 μM). (C) MCF 10A and MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, and spheroid formation was assessed at days 10 and 21. Representative photographs are shown. (D) MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, in the presence of rottlerin (5 μM), or BJE6-106 (1 μM or 5 μM), or DMSO vehicle (Control). Tumor spheroids were enumerated at 10 days. Representative photographs are shown. (E) Spheroid numbers were normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%) and plotted. Error bars represent SEM. p values for comparison between vehicle and rottlerin or BJE6-106 effects on spheroid number were significant (p < 0.001).

Mentions: A CSC-like phenotype can be induced during epithelial-mesenchymal transition (EMT) in transformed cell lines. Transformation of the “normal” human mammary epithelial cell line MCF 10A and selection for a tumorigenic, metastatic phenotype in vivo produced the derivative line MCF 10C [53,54], which exhibits an EMT phenotype [60]. Cells of this malignant derivative also became ALDH + [61]. Transformation of these cells rendered them sensitive to rottlerin (Figure 5A) and to BJE6-106 (Figure 5B), compared to the parental MCF 10A line. The IC50 of rottlerin and BJE6-106 for the MCF 10C derivative was approximately 1 μM and 0.1 μM, respectively, at 72 hr, whereas the IC50 for the parental MCF 10A cells were >20 μM.


Protein kinase C-δ inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo.

Chen Z, Forman LW, Williams RM, Faller DV - BMC Cancer (2014)

Effects of PKCδ inhibitors on growth and spheroid formation in non-transformed and transformed human breast epithelial cells. MCF 10A cells and cells from the derived tumorigenic line MCF 10C (also called M3), were grown to 80% confluence in 96-well plates and then exposed to rottlerin at concentrations ranging from 1 to 20 μM (A) or to BJE6-106 at concentrations ranging from 0.1 to 20 μM (B). The corresponding equivalent volume of solvent (DMSO) was used as a vehicle control (Vehicle). After 24, 48 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control (vehicle) values were normalized to 100%. Error bars represent SEM. p values for comparison between vehicle and PKCδ inhibitors on MCF 10A cell number only reached significance (p < 0.05) at 48 hr at 20 μM for rottlerin, and at 1 μM for BJE6-106. In contrast, significant effects of the inhibitors on the MCF 10C cells were observed as early as 24 hr for rottlerin (at 5 μM) and for BJE6-106 (at 0.1 μM). (C) MCF 10A and MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, and spheroid formation was assessed at days 10 and 21. Representative photographs are shown. (D) MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, in the presence of rottlerin (5 μM), or BJE6-106 (1 μM or 5 μM), or DMSO vehicle (Control). Tumor spheroids were enumerated at 10 days. Representative photographs are shown. (E) Spheroid numbers were normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%) and plotted. Error bars represent SEM. p values for comparison between vehicle and rottlerin or BJE6-106 effects on spheroid number were significant (p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Effects of PKCδ inhibitors on growth and spheroid formation in non-transformed and transformed human breast epithelial cells. MCF 10A cells and cells from the derived tumorigenic line MCF 10C (also called M3), were grown to 80% confluence in 96-well plates and then exposed to rottlerin at concentrations ranging from 1 to 20 μM (A) or to BJE6-106 at concentrations ranging from 0.1 to 20 μM (B). The corresponding equivalent volume of solvent (DMSO) was used as a vehicle control (Vehicle). After 24, 48 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control (vehicle) values were normalized to 100%. Error bars represent SEM. p values for comparison between vehicle and PKCδ inhibitors on MCF 10A cell number only reached significance (p < 0.05) at 48 hr at 20 μM for rottlerin, and at 1 μM for BJE6-106. In contrast, significant effects of the inhibitors on the MCF 10C cells were observed as early as 24 hr for rottlerin (at 5 μM) and for BJE6-106 (at 0.1 μM). (C) MCF 10A and MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, and spheroid formation was assessed at days 10 and 21. Representative photographs are shown. (D) MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, in the presence of rottlerin (5 μM), or BJE6-106 (1 μM or 5 μM), or DMSO vehicle (Control). Tumor spheroids were enumerated at 10 days. Representative photographs are shown. (E) Spheroid numbers were normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%) and plotted. Error bars represent SEM. p values for comparison between vehicle and rottlerin or BJE6-106 effects on spheroid number were significant (p < 0.001).
Mentions: A CSC-like phenotype can be induced during epithelial-mesenchymal transition (EMT) in transformed cell lines. Transformation of the “normal” human mammary epithelial cell line MCF 10A and selection for a tumorigenic, metastatic phenotype in vivo produced the derivative line MCF 10C [53,54], which exhibits an EMT phenotype [60]. Cells of this malignant derivative also became ALDH + [61]. Transformation of these cells rendered them sensitive to rottlerin (Figure 5A) and to BJE6-106 (Figure 5B), compared to the parental MCF 10A line. The IC50 of rottlerin and BJE6-106 for the MCF 10C derivative was approximately 1 μM and 0.1 μM, respectively, at 72 hr, whereas the IC50 for the parental MCF 10A cells were >20 μM.

Bottom Line: Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture.PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours.Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Center, Boston University School of Medicine, K-712C, 72 E, Concord St,, Boston, MA 02118, USA. dfaller@bu.edu.

ABSTRACT

Background: A subpopulation of tumor cells with distinct stem-like properties (cancer stem-like cells, CSCs) may be responsible for tumor initiation, invasive growth, and possibly dissemination to distant organ sites. CSCs exhibit a spectrum of biological, biochemical, and molecular features that are consistent with a stem-like phenotype, including growth as non-adherent spheres (clonogenic potential), ability to form a new tumor in xenograft assays, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. PKCδ is a novel class serine/threonine kinase of the PKC family, and functions in a number of cellular activities including cell proliferation, survival or apoptosis. PKCδ has previously been validated as a synthetic lethal target in cancer cells of multiple types with aberrant activation of Ras signaling, using both genetic (shRNA and dominant-negative PKCδ mutants) and small molecule inhibitors. In contrast, PKCδ is not required for the proliferation or survival of normal cells, suggesting the potential tumor-specificity of a PKCδ-targeted approach.

Methods: shRNA knockdown was used validate PKCδ as a target in primary cancer stem cell lines and stem-like cells derived from human tumor cell lines, including breast, pancreatic, prostate and melanoma tumor cells. Novel and potent small molecule PKCδ inhibitors were employed in assays monitoring apoptosis, proliferation and clonogenic capacity of these cancer stem-like populations. Significant differences among data sets were determined using two-tailed Student's t tests or ANOVA.

Results: We demonstrate that CSC-like populations derived from multiple types of human primary tumors, from human cancer cell lines, and from transformed human cells, require PKCδ activity and are susceptible to agents which deplete PKCδ protein or activity. Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture. PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours. Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.

Conclusions: These findings suggest that the novel PKC isozyme PKCδ may represent a new molecular target for cancer stem cell populations.

Show MeSH
Related in: MedlinePlus