PIM (Proviral Insertion site in Moloney murine leukemia virus) proteins are a family of short-lived serine/threonine kinases that are upregulated in several human cancers (such as prostate cancer, acute myeloid leukemia and other hematopoietic malignancies). Usually, PIM kinase family are mainly responsible for enhancement of cell cycle, inhibition of apoptosis, activation of transcription, and cell homing/migration. These characteristics of PIM kinases lead their encoding genes (named Pim) to be potent carcinogenesis promoting genes and raise a great attention to researchers.
The PIM kinase family
The PIM kinase family contains three members: PIM-1, PIM-2 and PIM-3 with partially specific tissue expression but overlapping functions. Pim genes are located in different chromosomes in the mouse and human genome, as shown by black numbers and numbers in orange boxes, respectively (Figure 1). Though with partially tissue specific expression in the body, the amino acid of each kind of PIM kinase has shown a high level of homology. At the amino acid level, PIM-1 and PIM-3 are 71% identical, whereas PIM-1 and PIM-2 share 61% homology. Each Pim gene has six exons (shown blue in the figure 1) that give rise to the mRNA, and with alternative initiation of mRNA transcription, the Pim genes will produce a large family of PIM proteins with different molecular mass, but they all contain the highly conserved serine/threonine kinase domain (as shown in the white box in the protein structure of the figure 1).
Unlike the traditional protein kinase enzymes, PIM proteins are not regulated by membrane recruitment or phosphorylation, they are mainly controlled by the cell transcription. Pim-1 gene produce two isoforms of PIM protein with 34 KD and 44 KD by alternative initiation of Pim-1 mRNA. The 44 KD PIM-1 contains an N-terminal proline-rich motif that binds the ETK SH3 domain and is recruited to the plasma membrane. Three isoforms of PIM-2 proteins were produced by Pim-2 gene transcription and translation with the molecular weight of 34 KD, 37 KD, and 38KD. PIM-3 gene only produce one protein without isoforms. As said, they have many similarities in the amino acid level and have same functional enzyme site, it can be proposed that they maybe compensate for each other if a certain protein is loss in the cell.
PIM kinases are constitutively active without regulatory domain. So it has the function when it was stabilized in the cell. Signals inducing Pim genes expression are diverse and tissue dependent.
Figure 1 PIM genes, transcripts and proteins
Upstream signaling of PIM kinases
Functional PIM protein only lasted its activity less than 5 min, so the regulation of PIM protein is mainly dependent on the cell transcription, translation and the proteasome degradation. Pim genes generally display characteristics of primary response genes (PRGs) that are induced by the activation of transcription factors downstream of growth factor signaling pathways, such as the janus kinase and signal transducer and activator of transcription (JAK-STAT), and nuclear factor-κB (NF-κB; also known as NFKB1). Also it should be concerned that the recent observation in solid tumors has shown that PIM protein is elevated by hypoxia in a hypoxia-inducible factor1α (HIF1α)-independent manner.
Mitogenic stimuli induce Pim genes transcription as a response for the activation of JAK-STAT pathway. Cytokine receptors like growth factors (SCF, G-CSF and EGF)，interleukins (IL 2~11) activate the STAT proteins via the JAKs. The activation of Jaks leads to the STATs protein dimerization and nuclear translocation, wherein the STATs bind to specific promoter regions of the target genes and regulate their expression. For example, the STAT 3 and STAT 5 will bind to the promoter of the Pim-1 gene, and upregulate the Pim-1 gene expression. After the PIM was produced, it will enhance the cell cycle. On the other hand, it’ll also activate another protein named SOCS (suppressor of cytokine signalling) and prevent the activation of JAK-STAT pathway as a negative feedback. Shortly while, the PIM protein will go to the degradation period. There are also regulations in this period, the Protein phosphatase 2 (PP2A) will phosphorylate the PIM protein and lead to the ubiquitination dependent degradation by binding with the HSP70.
The NF-κB pathway was also thought to join in the PIM genes regulation, but the underlying mechanisms is not clear now, so do the hypoxia-induced elevation of PIM proteins.
Figure. 2 Upstream signaling of PIM kinase
Downstream signaling of PIM kinases
As a kinase protein, PIM phosphorylate a large set of substrates. The PIM-1 exhibits a strong preference for the substrates containing K/R-X-X-X-S/T-X motifs (X is neither basic nor a large hydrophobic residue). Protein-protein interaction studies have found many putative substrates of PIM kinase, including CBX3, CDC-25A, HSP90, HSP70, NFATC-1, NUMA, P21, SND-1, RELA, HP-1, PAP-1, SNX-6, and p100.
A consensus site that PIM phosphorylated was the cell cycle regulator P21. All the three PIM isoforms were found interacting with the P21 at the different binding site. Also, the CDC-25A is also in the cell cycle regulation, PIM kinase can physiologically activate CDC-25A and induced a c-Myc signaling in the upstream of the P21. Furthermore, PIM kinases appear to repress p27 transcription via phosphorylation and inactivation of FoxO1a and FoxO3a. These proteins are all essential in regulation of cell cycle.
Pro-survival activity of PIM kinases was shown in the PIM kinase overexpression cells. This phenomenon can be interpreted by the phosphorylation of BAD protein. Usually, dephosphorylated BAD forms a heterodimer with Bcl-2 and Bcl-xL. When BAD protein is phosphorylated, it forms the BAD heterodimer and leaves the Bcl-2 free. The free Bcl-2 can inhibit the Bax-triggered the apoptosis. Thus, the phosphorylation of BAD by PIM is anti-apoptosis.
PIM-2 has been shown to phosphorylate the ribosomal protein 4E-BP1, causing its dissociation from eukaryotic initiation factor 4E (eIF4E), and resulting in the activation of cap-dependent mRNA translation. Activity of PIM-2 kinase on this protein will finally affect the mTOR pathway and change the metabolism parameters.
Recent research of the P53 pathway has shown that endogenous PIM-1 and PIM-2 interact with endogenous Mdm2, which is an important negative regulator of the p53 tumor suppressor. CXCR4 was also regulated by PIM kinase and involved in the cancer cell invasion and metastasis.
Except for the functions discussed above. PIM kinase also affected the genomic instability. This activity is largely due to its association with the nuclear mitotic apparatus (NUMA) protein. It has been shown that checkpoint control is lost under PIM-1 overexpression, and as a consequence, cells with spindle abnormalities are not arrested in mitosis, resulting in polyploidy and multi-nucleation
Figure 3 Downstream signaling of PIM kinase
PIM kinases as a therapy target
PIM is highly expressed in the human cancer and joined in many cancer specific pathways. It raised PIM kinase as an active target for cancer drug discovery research. Glucose deprivation induced apoptosis under hypoxia is sensitized after dominant negative mutation of Pim-1 gene. Prevention of PIM function by the shRNA also decreases the cell growth capability and invasion. The most important thing is to develop a specific inhibitor of PIM-1 protein, since its known implications in tumorigenesis. The sarcomas arising from PIM kinase deficiency mice have shown a significantly reduced expression of proliferation markers, which suggests the inhibition of tumor growth. The protein level of 4E-BP1, GSK3β and pERK is decreased, these all contribute to the inhibition of tumor growth rate. PIM mediated chemoresistance of cancer cells through BAD inactivation and hypoxia-induced drug resistance which is another pivotal question remained to be solved. Inhibition of the activity of the transcription factor ETs1 in human pancreatic cancer cells impairs PIM-3 transcription and protein expression with concomitant reduction of BAD phosphorylation on s112 and increased apoptosis, which could be reversed by exogenous PIM-3 expression
PIM kinase inhibitors
More than 100 PIM inhibitors had been reported according to the literature. But few of them was tested in vitro or in vivo to prove its antitumor efficiency. Many PIM kinase inhibitors inhibit activity of all three isoforms, though no clear side effect reported, we still want to find some inhibitors that selectively target the PIM-1 kinase. A number of in vivo tests have shown that the inhibitor dose can easily reach the level required for the inhibition of all the PIM kinases. Moreover, inhibition of all PIM kinases maybe is an efficacious way to cure the cancer patients. Remarkably, the combination of PIM kinases inhibitor with other classical-target therapies seems promising in the cancer therapy.
SGI-1776 is a nonspecific inhibitor of PIM kinases at a nanomolar range. SGI-1776 treatment reduced cell viability and recovered the sensitivity to taxane-based therapies in chemo-resistant cells by inhibiting multidrug resistance 1 activity. Similar with the knock-down of PIM kinase, SGI-1766 treatment prevented the P-glycoprotein from degradation and enabled its glycosylation and cell surface expression. SGI-1766 treatment on the CLL cell line reduced the c-Myc-MCL-1 signaling, which will promote the apoptosis. In the phase I clinical trial, the evaluation of the compound was halted due to cardiac toxicity. CX-4945 is a dual inhibitor of PIM/CK protein, with IC50 values of 1 nM for CK2, 46 nM for PIM-1, 186 nM for PIM-2 kinase. In 2009, the phase I clinical trial was studied by oral administration of CX-4945 in patients with advaced solid tumors, breast cancer, inflammatory breast cancer or multiple myeloma. Of the patients on the twice-daily regimen, 17% were reported to maintain stable disease for more than 6 months, and 9% were reported to maintain stable disease for more than 12 months. The phase I study is still going on to test the safety and tolerability of CX-4945 in patients with multiple myeloma.
There are many compounds still on the way to be developed as potent drugs for the cancer therapy. The PIM kinases inhibitors, combined with other signaling pathway regulators, will play an important role in the future cancer therapy.
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