Inhibition Of Polo-like Kinase 1 Selectively Promotes Regression Of RAS Mutant Bladder Cancer And Establishment Of Patient-Derived Xenografts Of Bladder Cancer

1. Inhibition of polo-like kinase 1 selectively promotes regression of RAS mutant urinary bladder cancerBladder cancer (BCa) is a common malignant tumor in urinary system. It is a heterogeneous disease, and more than 90% of the tumors are transitional cell carcinoma. The clinical and pathological studies indicate that the BCa can be initiated by more than two separate mechanisms, which makes the target therapy of bladder tumor as a great challenge. It has been reported that RAS mutation is sufficient to induce BCa. However, direct inhibiting Ras protein was not successful to date. Recently, synthetic lethal RNAi screening have identified several genes involved in the synthetic lethal interaction with oncogene RAS, and PLK1 is the first gene identified in this process. However, whether inhibiton of Plkl could effectively suppress RAS mutant Bca is still not clear.In this study, we found that inhibition of plkl selectively promotes regression of RAS mutant urinary. Blockade of Plkl activity either by siRNA or specific chemical inhibitors (BI-2536 and BI-6727) could lead to significant cytoxicity to RAS mutant bladder cancer cells when compared with the wild type ones (P<0.01). Comparing to RAS wild type BIU-87 cells, inhibition of Plkl also resulted in cell cycle arrest at G2/M in RAS mutant 5637 cells, followed by excessive apoptosis. We further explored the mechanism and found that Plkl suppression via specific inhibitors decreased p-ERK and p-AKT levels in RAS mutant bladder cancer cells. However, this did not occur in RAS wild type bladder cancer cells. In vivo xenograft mouse model bearing RAS mutant bladder cancer, BI-2536 (40 mg/kg/w, intravenous injection,22 day) evidently inhibited growth and tumor weight (n=8), without observable toxicity. Immunohistochemistry revealed that the decreased expression of Ki-67, p-ERK and p-AKT in those treated tumors. Our finding shows that inhibition of Plkl selectively inhibits RAS mutant bladder tumor by interfering with Ras downstream ERK and AKT pathway, providing a new strategy for the treatment of RAS mutant bladder cancer.2. Establishment of Patient-Derived Xenografts of Bladder CancerAnimal models play an important role not only in the tumorigenesis mechanism research, but also in new drug discovery. Most of the cancer xenograft models are derived from tumor cell lines, however, they do not sufficiently represent clinical cancer characteristics. Since the microenvironment has a critical impact on the growth of tumor, it’s important to choose appropriate animal model in cancer research.Recently, a few groups have developed more relevant models based on xenografting primary human tumor tissue in 5-week-old male immunodeficient mice (BALB/c-nu).Patient-derived tumor tissue xenograft models retain architecture, morphology,and molecular signatures similar to the original cancers. Therefore, the aim of our study was to develop xenograft model of BCa using patient-derived tumor tissues with heterogeneous clinico-pathological features, which would be useful for investigating BCa biology and developing new anticancer therapeutics.Written informed consent was obtained from each patient.In this study we cooperate with the Urology department of Changhai Hospital. The fresh human bladder tumor specimens were obtained at the time of surgery from 21 heterogeneous BCa patients.Surgical tumor samples were cut into pieces and injected subcutaneously into the immunodeficient mice. Mice were observed daily for tumor growth. "Sufficient tumor growth" was defined as the original tumor volume of 500 mm3, and then tumors were removed and passaged several times in nude mice (designated P1 to P5).From these 21 samples,1 tumor xenografts has successfully passaged to P3. By mutational analysis, we found that the 12th patient tumor had no RAS mutation in codon 12,13,16. However, molecular identification needs to be further developed. In conclusion, our BCa xenograft models derived from patient tumor tissue is expected to be useful for studying the heterogeneity of the tumor populations in bladder cancer and for evaluating new treatments.