The
26S proteasome is a
proteolytic enzyme found in both cytoplasm and nucleus. In this study, we examined the differential expression of
proteasome inhibitor bortezomib-induced
proteins in p53-deficient 4T1 cells. It was found that
GRP78 and TCEB2 were over-expressed in response to treatment with
bortezomib for 24h. Next, we analyzed the expression of intracellular
proteins in response to treatment with 100nM
bortezomib for 24h by label-free LC-MS/MS. These analyses showed that Hsp70, the
26S proteasome non-
ATPase regulatory subunit 14 and sequestosome 1 were increased at least 2 fold in p53-deficient 4T1 cells. The
proteins identified by label-free LC-MS/MS were then analyzed by Ingenuity Pathway Analysis (IPA) Tool to determine
biological networks affected by inhibition of the
26S proteasome. The analysis results showed that post-translational modifications, protein folding, DNA replication, energy production and
nucleic acid metabolism were found to be among the top functions affected by the
26S proteasome inhibition. The
biological network analysis indicated that
ubiquitin may be the central regulator of the pathways modulated after
bortezomib-treatment. Further investigation of the mechanism of the
proteins modulated in response to the proteasomal inhibition may lead to the design of more effective and novel therapeutic strategies for
cancer.
BIOLOGICAL SIGNIFICANCE: Although the
proteasome inhibitor bortezomib is approved and used for the treatment of human
cancer (
multiple myeloma), the mechanism of action is not entirely understood. A number of studies showed that
proteasome inhibitors induced apoptosis through upregulation of
tumor suppressor protein p53. However, the role of
tumor suppressor protein p53 in
bortezomib-induced apoptosis is controversial and not well-understood. The
tumor suppressor p53 is mutated in at least 50% of human
cancers and is strongly induced by proteasomal inhibition. Some also reported that the
proteasome inhibitor can induce apoptosis in a p53-independent manner. Also, it is reported that Noxa, a target of p53, is induced in response to proteasomal inhibition in a p53-independent manner. However, we have also previously reported that neither Puma nor Noxa are induced by proteasomal inhibition in p53-null 4T1
breast cancer cells, which is commonly used for in vivo
breast cancer tumor models. The current results provided additional targets of
proteasome inhibitor bortezomib and may therefore help in understanding the p53-independent mechanism of apoptosis induction by
proteasome inhibitors. In addition, the results presented in this current study report for the first time that proteasomal subunit Psmd14, anti-apoptotic
GRP78,
anti apoptotic protein Card10, Dffb,
Traf3 and Trp53bp2 are regulated and overexpressed in response to
proteasome inhibitor bortezomib in p53-deficient 4T1 cells. Therefore, novel therapeutic strategies targeting these anti-apoptotic or
pro-apoptotic proteins as well as inhibiting the
proteasome simultaneously may be more effective against
cancer cells. The
proteins identified here present new avenues for the development of anti-
cancer drugs.