< Back to previous page

Project

Insights into the pathways implicated in the addiction of cancer B-cells to Bcl-2 at the endoplasmic reticulum

The anti-apoptotic Bcl-2 protein is upregulated in many malignancies, including B-cell cancers such as diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), as this pro-survival protein functions as a negative regulator of programmed cell death or apoptosis. Bcl-2 inhibits Ca2+-mediated apoptotic cell death by suppressing toxic, pro-apoptotic Ca2+ release from the endoplasmic reticulum (ER), which is the main intracellular Ca2+ store. This is achieved by inhibiting the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), a Ca2+ channel located in the ER membrane that releases Ca2+ upon stimulation by its ligand IP3. A cell-permeable peptide tool, called BIRD-2, was developed to target cancer cells that rely on ER-localized Bcl-2 to hamper toxic, IP3R-mediated Ca2+-signaling events. BIRD-2 functions as a competitive inhibitor of the interaction between Bcl-2 and the IP3R channel, thereby reversing Bcl-2-mediated inhibition of the IP3R. Interestingly, BIRD-2 kills Bcl-2-dependent cancer cells, including DLBCL and CLL, by provoking spontaneous, pro-apoptotic Ca2+-signaling events. Moreover, we showed that the sensitivity of DLBCL cells to BIRD-2 correlates with the expression levels of IP3R2, which is the IP3R isoform with the highest sensitivity towards its ligand IP3. Thus, DLBCL cells with high IP3R2-expression levels respond well to BIRD-2, while DLBCL cells with low IP3R2 levels are rather resistant to the peptide. Although these recent studies expanded our understanding of how BIRD-2 kills Bcl-2-dependent cancer cells, it is clear that several other biological pathways contribute to the apoptotic sensitivity of tumor cells to the Bcl-2 antagonist. Therefore, the general aim of this project is to identify additional biological mechanisms that underlie the addiction of certain types of cancer cells to Bcl-2’s pro-survival function at the ER. In brief, we investigated the contribution of constitutive IP3 signaling, store-operated Ca2+ entry (SOCE), and hyperactive protein kinase B (PKB)/Akt signaling to cell death in response to BIRD-2.

First, we showed that constitutive IP3 signaling, occurring downstream of the B-cell receptor, is essential for the survival of B-cell cancers, since pharmacological inhibition of phospholipase C (PLC), which catalyzes the formation of IP3, caused cell death in B-cell cancers. However, mild PLC inhibition protected against BIRD-2-induced apoptosis in DLBCL and CLL, suggesting that pro-survival IP3 signaling is switched into pro-death signaling by abolishing Bcl-2’s inhibitory action on the IP3R with BIRD-2. These results indicate that constitutive IP3 signaling, besides high IP3R2-expression levels, is an important determinant that underlies the addiction of cancer cells to Bcl-2 at the ER Ca2+ stores.

Next, we showed that BIRD-2-induced apoptosis depends on both the ER Ca2+-store content and extracellular Ca2+, but not on Ca2+ influx through SOCE, in DLBCL cancer cells. We hypothesized that BIRD-2 treatment activates SOCE, an important Ca2+-influx pathway activated upon ER store depletion, since BIRD-2 triggers apoptotic cell death through excessive Ca2+ release from the ER. However, SOCE inhibition affected neither the Ca2+ response nor cell death provoked by BIRD‑2 in DLBCL cells, indicating that SOCE is not activated upon BIRD-2 treatment and does not contribute to the BIRD-2-triggered cell death response. On the other hand, BIRD-2-provoked apoptosis was reduced by chelating Ca2+ present in the extracellular environment, indicating that extracellular Ca2+ plays a critical role in BIRD-2-induced cell death. Furthermore, depletion of the ER Ca2+ store also reduced apoptotic cell death triggered by BIRD-2. In summary, these results show that extracellular Ca2+ as well as the ER Ca2+-store content are critical determinants of BIRD-2-triggered apoptotic cell death, whereas SOCE is not activated by the Bcl-2 antagonist.

Finally, we examined whether hyperactive PKB/Akt signaling reduces the sensitivity of cancer cells to BIRD-2. Importantly, PKB/Akt suppresses pro-apoptotic Ca2+ signaling at the ER by modulating IP3R3 activity. Hence, hyperactive PKB/Akt signaling, which has been observed in DLBCL cancer cells, might decrease the BIRD-2 sensitivity of DLBCL cells that express high levels of IP3R3. Our results indicate that BIRD-2-induced cell death is increased by inhibiting PKB/Akt signaling in DLBCL. However, this is likely not due to the effects of PKB/Akt on IP3R3-mediated Ca2+ release from the ER, since IP3-induced Ca2+ release and the BIRD-2-provoked Ca2+ response were not increased upon PKB/Akt inhibition. In contrast, we showed that PKB/Akt inhibition disrupts the balance between pro- and anti-apoptotic Bcl-2-family members, since blocking PKB/Akt signaling decreased the expression levels of anti-apoptotic Mcl-1 while expression of the pro-apoptotic Bim protein was increased. Hence, the potentiation of BIRD-2-induced cell death upon inhibition of PKB/Akt signaling in DLBCL is likely the result of altered expression levels of Bcl-2-family members.

In conclusion, we obtained new insights into how intracellular Ca2+ dynamics control apoptotic cell death processes in biological systems by studying the pathways that contribute to BIRD-2-induced cell death in cancer cells that are addicted to Bcl-2’s pro-survival function at the ER. These findings will potentially contribute to the development of novel cell-death therapies for the treatment of Bcl-2-dependent tumors.

Date:1 Oct 2014 →  30 Sep 2018
Keywords:cancer B-cells to Bcl-2
Disciplines:Laboratory medicine, Palliative care and end-of-life care, Regenerative medicine, Other basic sciences, Other health sciences, Nursing, Other paramedical sciences, Other translational sciences, Other medical and health sciences
Project type:PhD project