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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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Related in: MedlinePlus

Effects of PKCδ inhibitors on human tumor cell spheroid formation. (A) Hs578T and MCF7 were plated under adherent or non-adherent conditions. Tumor spheroids and adherent cells were collected at 96 hr, stained for CD24 and CD44, and analyzed by flow cytometry. (B) Hs578T, MCF7, breast cancer stem cells (BCSC) and pancreatic cancer stem cells (PCSC) were plated in tumor spheroid media, in the presence of rottlerin, BJE6-106, or DMSO (Control). Tumor spheroids were enumerated at 96 hr, and normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%). p values for comparison between vehicle and rottlerin or BJE6-106 effects were significant (p≤0.001). Photographs are of representative areas of the culture plates. (C) MCF7 cells were exposed BJE6-106 or to rottlerin at the indicated concentrations. 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 values were normalized to 100%. p values for comparison between vehicle and rottlerin effects on cell number at 24 hr reached significance at 5 μM, and for BJE6-106 at 0.5 μM (p ≤ 0.02), and were significant for all concentrations tested at 48 and 72 hr time points. (D) Hs578T cells were exposed to vehicle or BJE6-106 (1 μM) for 6, 12, 24, 48 or 96 hr. Viable cells were enumerated and re-plated in media without BJE6-206, and spheroid numbers were quantitated 96 hr later. p values for comparison between vehicle and BJE6-106 effects on spheroid number were significant after 6 hr of exposure (p≤0.02), and remained significant at all time points thereafter. Error bars represent SEM.
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Figure 4: Effects of PKCδ inhibitors on human tumor cell spheroid formation. (A) Hs578T and MCF7 were plated under adherent or non-adherent conditions. Tumor spheroids and adherent cells were collected at 96 hr, stained for CD24 and CD44, and analyzed by flow cytometry. (B) Hs578T, MCF7, breast cancer stem cells (BCSC) and pancreatic cancer stem cells (PCSC) were plated in tumor spheroid media, in the presence of rottlerin, BJE6-106, or DMSO (Control). Tumor spheroids were enumerated at 96 hr, and normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%). p values for comparison between vehicle and rottlerin or BJE6-106 effects were significant (p≤0.001). Photographs are of representative areas of the culture plates. (C) MCF7 cells were exposed BJE6-106 or to rottlerin at the indicated concentrations. 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 values were normalized to 100%. p values for comparison between vehicle and rottlerin effects on cell number at 24 hr reached significance at 5 μM, and for BJE6-106 at 0.5 μM (p ≤ 0.02), and were significant for all concentrations tested at 48 and 72 hr time points. (D) Hs578T cells were exposed to vehicle or BJE6-106 (1 μM) for 6, 12, 24, 48 or 96 hr. Viable cells were enumerated and re-plated in media without BJE6-206, and spheroid numbers were quantitated 96 hr later. p values for comparison between vehicle and BJE6-106 effects on spheroid number were significant after 6 hr of exposure (p≤0.02), and remained significant at all time points thereafter. Error bars represent SEM.

Mentions: Sphere formation assays, which have been commonly used to identify and purify normal and malignant stem cells, were used to select a “CSC-like population” from established human breast cancer cell lines Hs578T, MDA231 and MCF7. A subpopulation of these cell lines could grow as non-adherent spheres and be continuously propagated in a defined serum-free medium in vitro. Flow cytometry and immunofluorescence analysis indicated that sphere-derived cells from cell lines contained a much larger proportion of cells expressing CD44, a candidate surface marker of breast cancer stem cells, and/or a smaller proportion of cells expressing the non-stem cell marker CD24, compared with adherent cells (Figure 4A). The frequency of spheroid formation relative to input cell number was low for the tumor cell lines (≤2-3%), as expected. In contrast, spheroid formation from the cultures of primary PCSC or primary breast cancer stem cells (BCSC) was much more efficient (45% and 53%, respectively). As expected, the CD24/CD44 profiles of cells in the spheres derived from the primary PCSC and BCSC did not differ from the adherent cells (not shown).


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 human tumor cell spheroid formation. (A) Hs578T and MCF7 were plated under adherent or non-adherent conditions. Tumor spheroids and adherent cells were collected at 96 hr, stained for CD24 and CD44, and analyzed by flow cytometry. (B) Hs578T, MCF7, breast cancer stem cells (BCSC) and pancreatic cancer stem cells (PCSC) were plated in tumor spheroid media, in the presence of rottlerin, BJE6-106, or DMSO (Control). Tumor spheroids were enumerated at 96 hr, and normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%). p values for comparison between vehicle and rottlerin or BJE6-106 effects were significant (p≤0.001). Photographs are of representative areas of the culture plates. (C) MCF7 cells were exposed BJE6-106 or to rottlerin at the indicated concentrations. 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 values were normalized to 100%. p values for comparison between vehicle and rottlerin effects on cell number at 24 hr reached significance at 5 μM, and for BJE6-106 at 0.5 μM (p ≤ 0.02), and were significant for all concentrations tested at 48 and 72 hr time points. (D) Hs578T cells were exposed to vehicle or BJE6-106 (1 μM) for 6, 12, 24, 48 or 96 hr. Viable cells were enumerated and re-plated in media without BJE6-206, and spheroid numbers were quantitated 96 hr later. p values for comparison between vehicle and BJE6-106 effects on spheroid number were significant after 6 hr of exposure (p≤0.02), and remained significant at all time points thereafter. Error bars represent SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3927586&req=5

Figure 4: Effects of PKCδ inhibitors on human tumor cell spheroid formation. (A) Hs578T and MCF7 were plated under adherent or non-adherent conditions. Tumor spheroids and adherent cells were collected at 96 hr, stained for CD24 and CD44, and analyzed by flow cytometry. (B) Hs578T, MCF7, breast cancer stem cells (BCSC) and pancreatic cancer stem cells (PCSC) were plated in tumor spheroid media, in the presence of rottlerin, BJE6-106, or DMSO (Control). Tumor spheroids were enumerated at 96 hr, and normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%). p values for comparison between vehicle and rottlerin or BJE6-106 effects were significant (p≤0.001). Photographs are of representative areas of the culture plates. (C) MCF7 cells were exposed BJE6-106 or to rottlerin at the indicated concentrations. 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 values were normalized to 100%. p values for comparison between vehicle and rottlerin effects on cell number at 24 hr reached significance at 5 μM, and for BJE6-106 at 0.5 μM (p ≤ 0.02), and were significant for all concentrations tested at 48 and 72 hr time points. (D) Hs578T cells were exposed to vehicle or BJE6-106 (1 μM) for 6, 12, 24, 48 or 96 hr. Viable cells were enumerated and re-plated in media without BJE6-206, and spheroid numbers were quantitated 96 hr later. p values for comparison between vehicle and BJE6-106 effects on spheroid number were significant after 6 hr of exposure (p≤0.02), and remained significant at all time points thereafter. Error bars represent SEM.
Mentions: Sphere formation assays, which have been commonly used to identify and purify normal and malignant stem cells, were used to select a “CSC-like population” from established human breast cancer cell lines Hs578T, MDA231 and MCF7. A subpopulation of these cell lines could grow as non-adherent spheres and be continuously propagated in a defined serum-free medium in vitro. Flow cytometry and immunofluorescence analysis indicated that sphere-derived cells from cell lines contained a much larger proportion of cells expressing CD44, a candidate surface marker of breast cancer stem cells, and/or a smaller proportion of cells expressing the non-stem cell marker CD24, compared with adherent cells (Figure 4A). The frequency of spheroid formation relative to input cell number was low for the tumor cell lines (≤2-3%), as expected. In contrast, spheroid formation from the cultures of primary PCSC or primary breast cancer stem cells (BCSC) was much more efficient (45% and 53%, respectively). As expected, the CD24/CD44 profiles of cells in the spheres derived from the primary PCSC and BCSC did not differ from the adherent cells (not shown).

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