PIP5K1α is Required for Promoting Tumor Progression in Castration-Resistant Prostate Cancer
Introduction
Prostate cancer (PCa) at the advanced stages often progress to a state known as castration-resistant prostate cancer (CRPC). Patients with CRPC will inevitably develop metastatic diseases especially bone metastasis. Currently, no effective therapeutic agents are available for targeting metastatic CRPC . Thus, metastatic CRPC is an incurable disease with poor prognosis, and remains a major clinical challenge. During the development of CRPC, elevated level of androgen receptor (AR) is required and sufficient to confer androgen sensitive cells to castration resistant phenotypes. Modulated expression of co-factors of AR is one of the mechanisms that cause altered AR expression, leading to increased survival and invasiveness of PCa under the castration-resistant condition. Better understanding of the AR-dependent mechanisms including: 1) constitutively active AR and AR co-factors, 2) inappropriate restoration of AR and AR co-factors, or 3) indirect AR activation, is important for designing new therapeutic interventions to treat metastatic CRPC.
We have previously identified a lipid kinase phosphatidylinositol 4-phosphate 5 kinase (PIP5K1α) as an important co-factor of AR to activate transcription of AR target genes for prostate cancer cell proliferation and survival. Further, PIP5K1α acts together with matrix metalloproteinases 9 (MMP9) directly on AR to cooperatively promote angiogenesis and invasiveness of PCa. A previous study of ours using large patient cohorts has shown that PIP5K1α is highly expressed in primary PCa and is associated with poor PCa patient outcome . Moreover, elevated level of PIP5K1α significantly correlates with AR in primary PCa and metastatic lesions . Overexpression of PIP5K1α promotes tumor growth and invasiveness by increasing the activity of PI3K/AKT in mouse xenograft models.
Abnormal expression of AR and PIP5K1α/AKT pathways cooperatively contribute to growth, survival and invasiveness in various types of metastatic cancer. Given that PIP5K1α is a predominant kinase to produce phosphatidylinositol 4,5-trisphosphate PI(4,5)P2 (PIP2) for the activation of PI3K/AKT pathways, this implicates that PIP5K1α is a key player of the signaling cascades that promote cancer cell proliferation, survival and invasiveness. However, what specific region(s) of PIP5K1α are required to promote these biological events is not known.
It is an unmet need to develop novel therapeutic agents that can effectively target AR pathways in metastatic CRPC. Several novel approaches to inhibit interactions between AR and its co-factors by using peptidomimetics have been tested to inhibit the activity of both AR and its co-factors in PCa cell lines and xenograft mice, which show promising results. We have discovered a selective PIP5K1α inhibitor, ISA-2011B, that inhibits PIP5K1α kinase activity and blocks its downstream PI3K/AKT phosphorylation, leading to reduced growth and invasion of PCa in cell lines and xenograft mouse models . Elevated expression of AR was significantly down-regulated in PCa cells by using this PIP5K1α inhibitor. Consistently, the inhibitory effect of ISA-2011B on PCa is accompanied with its ability to significantly down-regulate the elevated expression of AR, MMP9 and VEGFR2. Further, inhibition of PIP5K1α using ISA-2011B led to proteasome-dependent degradation of both AR and AR-V7 proteins, which sensitizes resistant cell line 22RV1 to become responsive to enzalutamide treatment. However, the mechanism underlying the interaction between PIP5K1α and AR leading to the progression of CRPC remains to be investigated .
We elucidated the role of PIP5K1α and its molecular action in tumor growth and invasion by using C4-2 cells and DU145 cells. We characterized a gene edited CRPC cell line C4-2 that expressed an N-terminally deleted PIP5K1α. We elucidated the role of the N-terminal domain of PIP5K1α in growth, survival and invasion of CRPC in in vitro and in vivo models. We further investigated the underlying molecular mechanisms associated with the full-length PIP5K1α and its N-terminal sub-domain in regulation of AR, CDK1, MMP9 and their downstream factors that are involved in growth, survival and invasion of CRPC cells. We finally confirmed that PIP5K1α is an intriguing drug target, and its inhibitor ISA-2011B has a great potential as a targeted drug candidate for the treatment of CRPC.
Material and Methods (urval)
Holomonitor Analysis
The digital HoloMonitor® M4 live-cell imaging system and Hstudio™ M4 software (PHI AB, Lund, Sweden) were applied for measuring cell morphological changes and motility in response to drug treatment. The HoloMonitor is placed in an incubator at 37 °C with 5% CO2. For cell imaging, 0.3 × 106 cells were plated in the 6-well plate. The images of live cells were captured every 10 min during the entire treatment period of 48 h. For spheroid imaging, the spheroids were firstly prepared as described under the former “Tumor Spheroid assay” section, then was transferred to a 96-well plate, and the image was taken by HoloMonitor.
 |
| (G) The digital Holomonitor M4 live-cell imaging system was applied to capture the tumor spheroids derived from C4-2 cells transfected with Ctrl-siRNA or PIP5K1A-siRNA. Representative images of tumor spheroids are shown in the left panel. The quantification of the counts of tumor spheroids from each group is shown in the right panel. |
(D) Representative live-cell images from the digital
Holomonitor M4 microscope shows the morphological changes in C4-2 cells
that were treated with vehicle control (Ctrl) or ISA-2011B for 48 h.
Representative images of tumor spheroids derived from cells treated with
vehicle control (Ctrl) or ISA-2011B were taken by using a light
microscope in (E) and the Digital Holomonitor M4 microscope (F).
In addition, live cell holomonitor imaging analysis showed that ISA-2011B treatment led to a dramatic reduction in cell volume and motility of C4-2 cells (Figure 3D).
(K) Representative images of the tumor spheroids were taken using the digital Holomonitor M4.
Slutsatserna man drar av denna stora och komplexa studie beskriver man i slutordet:
Inga kommentarer:
Skicka en kommentar