In the present study we describe the effects of NF-κB inhibition by EriB on the OCSCs. We demonstrated that EriB, by blocking NF-κB activity, is able to suppress OCSC-mediated inflammation through the inhibition of cytokine production. More importantly, we show that it promotes OCSC death, therefore depleting the tumor of the possible source of tumor repair and recurrence.
CSCs or tumor-initiating cells represent a population of the tumor, which are chemoresistant and can undergo both processes of self-renewal and differentiation 11, 29, 30
. These cells can therefore survive chemotherapy, and undergo self-renewal and differentiation to rebuild the tumor, thus leading to disease recurrence 31
. Targeting these cells is thus pivotal for the prevention of cancer recurrence and metastasis and the improvement of patient survival.
We previously showed chemoresistance and differentiation capacity to be some key features of the OCSCs 16, 24
. Thus it is possible that these cells are the source of EOC recurrence. Further studies showed that another remarkable characteristic of the OCSCs is constitutive NF-κB activity, which can be enhanced by TNFα ligation 9, 11
. Moreover, these cells have a functional TLR4 pathway which can also contribute to enhanced NF-κB activity and cytokine production 32
. Therefore, in addition to being the possible source of recurrence, OCSCs may also be the source of a chronic inflammatory microenvironment.
Acute inflammation represents a critical signal to adult stem cells to initiate the process of tissue repair, which involves self-renewal (maintaining the pool of stem cells) and tissue renewal (differentiation on tissue specific cells) 7, 33
. In this setting, the source of the inflammatory signals has been associated with immune infiltrates, primarily macrophages, which respond to tissue damage by differentiating into the M2 type and secreting pro-inflammatory cytokines and chemokines 34–36
. Chronic inflammation on the other hand, may have detrimental effects by directly affecting abnormal progenitor cells and promoting tumorigenicity. Over two decades ago, Dvorak and co-authors described the tumor as an unhealed wound that produces a continuous source of inflammatory mediators (cytokines and chemokines) 37
. This inflammatory environment promotes cell survival, enhances cell proliferation and may even affect anti-tumoral immunologic response. Therefore the demonstration that OCSCs can create a chronic inflammatory microenvironment through the NF-κB pathway, provided us with a specific target for treatment.
We found that EriB is able to inhibit NF-κB activity, followed by suppression of cytokine production. This observation is relevant since cytokines are important for the process of tissue renewal, and in the case of the tumor, tumor recurrence. Targeting this pathway may therefore allow prevention of recurrence and may also affect the protumoral immunologic environment.
Interestingly, targeting the NF-κB pathway in the OCSCs induced apoptosis. These findings are in accordance with the role of NF-κB as a regulator of anti-apoptotic genes such as XIAP. Treatment with EriB induces a significant decrease in XIAP, which can then remove the inhibitory effects on the caspases. XIAP has been shown to be a central regulator of cell survival in EOC and major attempts have been made, without much success, to develop XIAP inhibitors 38–41
We also found that inhibition of NF-κB can reverse the resistance of OCSCs to TNFα and FasL. TNFα has been shown to promote, in some types of tumors, cell growth instead of apoptosis 42
. We previously demonstrated that while TNFα can induce cell death in the mOCCs, it can enhance growth of the OCSCs 10
. This pro-apoptotic effect of EriB is not only associated with TNFα but also with FasL, another membranal receptor. Like TNFα, FasL is also ineffective in inducing cell death in the OCSCs. However, pre-treatment with EriB is able to sensitize OCSCs to FasL.
Finally we evaluated how EriB inhibits NF-κB activity. Our data demonstrated that EriB decreased the levels of nuclear p65 in the OCSCs, both at the baseline level and after TNFα treatment. Since NF-κB is a transcription factor, the inhibition of its nuclear translocation could explain the decrease in cytokines observed in the OCSCs after EriB treatment.
The nuclear translocation of p65 depends on its association with IκB 43
. Upon its phosphorylation of IκB, it gets degraded and this allows the dissociation and nuclear translocation of p65. As stated previously, OCSCs have constitutive NF-κB activity and the IκB levels in these cells cycles over a 24h period. We found the inhibition of this cycle after the OCSCs were treated with EriB. This suggests that EriB is able to prevent IκB degradation.
In summary, we report a novel compound, which induces death in the OCSCs by inhibiting the NFκB pathway – a pathway which is highly activated in the OCSCs cells. Identifying and understanding the pathways in the OCSCs that can confer survival, resistance to apoptosis, and capacity for repair is relevant to improve survival of ovarian cancer patients.