In this study, we investigated the expression levels of all six antiapoptotic Bcl-2 subfamily members at the mRNA level in 68 cancer cell lines across nine basic tissue types using qPCR techniques. We then measured the ability of chemical Bcl-2 inhibitors to induce cell death in 36 of the studied lines. In seven of nine tissue types, Mcl-1 was expressed at the highest levels among the family members, comprising from 40–60% of Bcl-2 subfamily mRNA present. Bcl-2 was found to be prominently expressed in leukemia/lymphoma lines but not in solid tumor cell lines. Melanoma cell lines and the melanocyte control sample all significantly overexpressed Bfl-1 at levels 9–150-fold higher than in HDF reference cells. We then studied the ability of three previously described Bcl-2 subfamily inhibitors26, 30
to induce cell death in a collection of the 36 cell lines screened for mRNA copy number.
Although we acknowledge that mRNA levels cannot definitely predict protein levels present in a cell, prior studies of the antiapoptotic Bcl-2 subfamily members protein levels correlate well with our findings.21, 22, 23
It has also been shown that constant turnover of both protein and mRNA levels are integral parts of Bcl-2 regulation.34
Moreover, to investigate the ability of the qPCR data to predict the efficacy of Bcl-2 antagonists, we tested three chemical Bcl-2 inhibitors (ABT-737, 8Q, and 8J) against a selection of the tumor cell lines studied. In support of the predictive nature of the mRNA studies here presented, we found an inverse correlation (r
<0.0001) between ABT-737-induced LD50
values and bcl-2
mRNA copy number (). Furthermore, this correlation was modulated by Mcl-1 (r
=−0.8081) and total antiapoptotic Bcl-2 subfamily (r
=−0.8135) mRNA levels, consistent with prior preclinical studies using Mcl-1 knockdown approaches.20
values for ABT-737 did not correlate with either bcl-xL
mRNA expression despite biochemical analysis showing that ABT-737 has potent nanomolar affinity to these proteins. This result suggests that either ABT-737 binding to Bcl-xL
or Bcl-W does not result in inhibition of their antiapoptotic functionality, or these two proteins are not critical for the survival of the tumor cell lines studied in routine cultures. However, it must be noted that therapies targeting antiapoptotic Bcl-2 family proteins may sensitize tumor cells to apoptotic stimuli, such as chemotherapy or radiation, without displaying single-agent cytotoxic activity.
A similar, though much weaker correlation was identified between observed LD50 values for compound 8J and calculated bcl-2 mRNA copy number (r=−0.4143, P=0.01). Given the wider activity across all subfamily members and the small range of observed LD50, improved information on the individual importance of each Bcl-2 subfamily member responsible for such a pan-active inhibitor would require either the discovery of a highly responsive cell line, development of improved Apogossypol derivatives with sub-micromolar affinities for individual Bcl-2 subfamily members, or intense studies of single cell lines while knocking out different subfamily members using siRNA techniques. Such multivariate analysis could prove very informative in the further optimization of pan-active Bcl-2 subfamily inhibitors. In contrast, a more broad-spectrum Apogossypol derivative, 8Q, which shows micromolar affinity against all six Bcl-2 subfamily members did not have an observable correlation between its LD50 values and the mRNA levels of any single subfamily member. This is likely due to the small range of LD50 values observed in the tested cell lines. Nevertheless, the correlations identified, especially for ABT-737, demonstrate the predictive nature of the reported qPCR studies.
Our data support and supplement to some extent results reported in prior studies focused on single cancer tissue types that looked at only a subset of the subfamily members (e.g. Bcl-2, Bcl-xL
, or Mcl-1) assessing relative protein levels using IHC methods. These studies found that for all cancer types, except leukemia, Mcl-1 is upregulated and can often be associated with some metric of cancer progression or poor patient prognosis.10, 15, 17
Our data agree with these studies in as much as mcl-1
mRNA is prominently expressed in most tumor cell lines. In this regard, it is typical that only the most aggressive cancers can be established readily as cell lines that can be easily passaged long-term in culture. Thus, the striking expression of mcl-1
mRNA observed in established tumor cell lines may be a reflection of their more aggressive character. In addition, siRNA studies targeting Mcl-1 have demonstrated that reduction of mcl-1
mRNA levels causes increased sensitivity to ABT-73735
and decreased resistance to anoikis.22
We must note that qPCR techniques are not able to differentiate among the posttranslational modifications of individual members of the Bcl-2 subfamily at the protein level. In this regard, it has been reported that significant stabilization or proteolysis of Bcl-2 and Mcl-1 protein may be facilitated by phosphorylation.36
Furthermore, the turnover of antiapoptotic Bcl-2 subfamily members has been reported to be dependent on several other proteins, including the levels of proapoptotic Bcl-2 subfamily members, the E3 ubiquitin ligase, MULE/LASU1, or individual caspases, to name a few.37
Although this is the case, we feel that by presenting a wide survey of the antiapoptotic Bcl-2 subfamily expression in cancer types this study will allow more dedicated investigation into these various control systems. A prior focused study of apoptosis gene expression in the NCI-60 cells lines only characterized the expression of Bcl-2 and Bcl-XL
It should also be noted that many of the prior studies were conducted with primary patient samples that are not readily available throughout the scientific community, nor in sufficient quantity to allow repeated probing for the discernment of LD50
values in the context of drug optimization programs. Thus, further development of the Bcl-2 field requires the analysis of available tumor cell lines, which serve as models to refine knowledge of biochemical signaling pathways and develop improved antiapoptotic Bcl-2 subfamily-targeted inhibitors. Owing to the large number of studies already conducted on the NCI-60 panel of cancer lines, we attempted to include as many of these cell lines as possible in our analysis.
As a result of this study, we feel that the current difficulties of Bcl-2-targeted inhibitors in the clinic39
may be understood through our observations that the severe upregulation of Bcl-2/Bcl-xL
seems to be isolated to leukemia like cell lines and that in other cancer types a strategy targeted to Mcl-1 inhibition (or Mcl-1/Bfl-1 inhibition in the case of melanoma) may prove to be more successful. In addition, although targeting individual members of the antiapoptotic Bcl-2 subfamily may initially prove promising, recent studies with ABT-737 suggest that resistance to the compound is a consequence of the ability of cancer cells to adapt and express other Bcl-2 subfamily members.20
Thus, the development of pan-active Bcl-2 inhibitors may lead to more successful therapeutic outcomes to be achieved. Our data illustrates that even within a cancer tissue type significant deviations may be seen between cell lines. Taken together, these data argue clinical trials seeking to develop Bcl-2 inhibitors should minimally include assessment of the expression of all six antiapoptotic Bcl-2 subfamily members so that the activity profile of compounds can be connected with the repertoire of Bcl-2 family members present within the tumor of individual patients.