While investigating prostate cancer stem cell properties, we found that commonly used human prostate cancer cell lines could be induced to differentiate into osteoblast-like () or adipocyte-like cells (). These findings are in concordance with clinical observations, in which prostate tumor cells often express osteoblastic markers (12
). The results are also in agreement with previous studies where LNCaP and C4-2 prostate cancer cells were induced to express osteoblastic phenotypes (14
). In this study, we additionally demonstrated that the tumor cells could express adipogenic markers as well. The brown adipocyte-specific UCP1, for example, was abnormally enhanced in most primary tumors and all bone metastases (), suggesting that in clinical prostate cancer, certain tumor cells may harbor mesenchymal stem cell properties as well. Osteoblastic and adipogenic differentiation are unique capabilities of bone marrow mesenchymal stem cells. Human prostate cancer cells thus harbor the bone marrow mesenchymal stem cell properties. The bone marrow is a unique microenvironment for mesenchymal stem cells, and tumor cells with bone marrow mesenchymal stem cell-like properties could have an advantage for homing to and colonizing bone. It would be intriguing to investigate whether these intrinsic properties are an underlying cause of the preferential metastasis of prostate cancer to bone.
This study showed that different prostate cancer cell lines have distinct potentials for lineage-specific differentiation. Cells of the LNCaP lineage appeared to have the potential to differentiate into osteoblast-like cells, while adipogenic differentiation was most prominent in the PC-3 lineage. It seemed that each prostate cancer cell line had limited differentiation potentials for the bone marrow mesenchymal stem cell lineage.
Though prostate cancer cells are known to have osteomimetic properties (12
), this study is the first to recognize their adipogenic potential. Subsequently, PC-3 lineaged cells and DU145 cells were used to characterize the phenomenon. These cells expressed a high level of PPARγ (), the master regulator of adipogenic differentiation (25
). Together with the expression of downstream adipogenic markers FABP4/aP2 and PLIN1, these assays confirmed the adipogenic stem cell property. Adipogenic induction resulted in morphologic changes similar to brown fat cells (, , and ). In concordance to the induced expression of brown adipocyte-specific UCP1 (), these results strongly suggested that prostate cancer cells had differentiated into brown fat-like cells. Moreover, this study characterized the abnormally enhanced UCP1 expression as a unique marker for prostate cancer progression and bone metastasis.
Unlike white fat cells, brown adipocytes consume energy rather than serving as energy storage (25
), while UCP1 protein uncouples oxidative phosphorylation from ATP synthesis to cause mitochondrial proton leak by dissipating energy as heat (34
). Intriguingly, increased expression of UCP1 has been postulated to be involved in cancer cachexia (35
). Further investigation into UCP1 may define the cause of cachexia during prostate cancer progression and metastasis.
This study revealed that the adipocyte-like cancer cells could reverse the differentiation program by retrodifferentiation, while the retrodifferentiated cells could be induced again to differentiate into adipocyte-like cells (). On the other hand, we determined that adipocyte differentiation in the mMSC appeared to be an irreversible process (data not shown). It seemed that prostate cancer stem cells harbored extraordinary mechanisms empowering retrodifferentiation, which is an ancient mechanism invoking apoptosis escape and cell cycle re-entry (37
). Considered as an approach to cellular rejuvenation and regeneration, retrodifferentiation has been explored as a treatment for degenerative and dystrophic diseases, but the underlying regulatory mechanism remains obscure. Retrodifferentiation is potentially a cause of prostate cancer recurrence, and the current study has established a model to illustrate its mechanism of molecular regulation.
Human prostate cancer cell lines responded differently to differentiation induction. Under adipogenic conditions, for instance, cells of the LNCaP lineage and DU145 cells responded slowly with limited lipid droplet production but sensitively with growth arrest. Cells of the PC-3 lineage showed opulent lipid droplets and acute apoptotic death. The mechanism underlying the differential sensibility to induced differentiation remains to be elucidated by comparative studies. Importantly, the adipogenic induction inhibited the growth of cancer cells () and most adipocyte-like cancer cells in the PC-3 lineage died through apoptosis (), implying that induced differentiation could be an effective therapeutic strategy. Induced differentiation has been used to treat stem cell malignancies by pushing immature cells to differentiate into a more mature state (38
), since mature cells have limited life spans and would perish eventually through programmed mechanisms (29
). Acute promyelocytic leukemia, for example, can be effectively treated with all-trans
retinoic acid, a specific inducer for immature blood cells to differentiate and die (40
). Further investigation is warranted to evaluate the adipogenic differentiation as a therapeutic modality for prostate cancer. In our study, the inducing agents for adipogenic differentiation were the glucocorticoid dexamethasone and the non-steroid anti-inflammatory drug indomethacin. Dexamethasone and indomethacin have been used as monotherapies for prostate cancer (41
), while their efficacy in combinatory use remains to be evaluated. We have observed synergistic effects of the combinatory use on PC-3 cell adipogenic differentiation. It would be interesting to determine whether an adipogenic differentiation therapy, in combination with novel strategies to block retrodifferentiation, can effectively inhibit prostate cancer recurrence and prevent metastasis.
It is paradoxical to notice that although in vitro UCP1 induction in prostate cancer cells, especially in the PC-3 lineage, was accompanied by growth inhibition and marked apoptosis (); progressively enhanced UCP1 expression was correlated with malignant status in clinical prostate cancer specimens (). Whether the UCP1 level is accompanied by growth inhibition and apoptosis in vivo remains to be determined. It becomes critical to simultaneously evaluate the adipogenic marker expression versus growth inhibition and apoptosis in individual clinical prostate cancer specimens. On the other hand, being a unique protein in mature brown fat cell mitochondria mediating energy dissipation, UCP1 may serve as a brown adipocyte lineage marker, but may not be integral to growth arrest and programmed death, which could be controlled by mechanisms independent of UCP1 protein function.
The study of prostate cancer stem cells is at an early stage, and how mesenchymal stem cell properties come into existence in prostate cancer cells is presently unclear. RWPE-1 and PrEC cells were used in this study to represent normal prostate epithelial cells, and these cells could not be induced to undergo either osteoblastic or adipogenic differentiation (), implying that the mesenchymal stem cell properties in the cancer cells were acquired abnormalities. This study revealed that a single prostate cancer cell line could have the stem cell properties of heterogeneous cell lineages. The extent of heterogeneity remains to be identified, but tumor cells might have opportunities to acquire the additional stem cell properties of multiple cell lineages during the chronic process of prostate cancer progression. Prostate cancer cells of the LNCaP lineage, for instance, could be induced to express neuroendocrine phenotypes, while clinical prostate cancer has been known for neuroendocrine differentiation (43
). Although adipogenic induction alone may promote differentiation-induced programmed death of the cancer cells that harbor adipogenic stem cell properties, further investigations have to be conducted to search and define other stem cell properties that could be exploited for prostate cancer differentiation therapy.