Cancer Letters

Cancer Letters

Volume 436, 1 November 2018, Pages 28-37
Cancer Letters

Original Articles
PTEN deficiency confers colorectal cancer cell resistance to dual inhibitors of FLT3 and aurora kinase A

https://doi.org/10.1016/j.canlet.2018.08.011Get rights and content

Highlights

  • A synthetic lethality screen identified mutant-PTEN colorectal cancer cell resistance to dual inhibitors of FLT3 and aurora kinase A (AURKA).

  • The dual inhibitors induced a dose-dependent, biphasic reactivation of AKT phosphorylation at Ser473 in mutant-PTEN cells.

  • AURKA inhibition leads to inhibition of AKT phosphorylation at Ser473, while co-inhibition of AURKA with FLT3 reactivates AKT in PTEN mutant cells.

  • AKT inhibition reversed the drug resistant phenotype in mutant-PTEN cells to the dual inhibitors.

Abstract

PTEN is a tumor suppressor found mutated in many cancers. From a synthetic lethality drug screen with PTEN-isogenic colorectal cancer cells, we found that mutant-PTEN cells were resistant to dual inhibitors of FLT3 and aurora kinase-A, including KW2449 and ENMD-2076. KW2449 significantly reduced the viability of wildtype-PTEN cells causing apoptosis, while little effect was observed in mutant-PTEN counterparts. Transcriptome profiling showed that members of PI3K-AKT signaling pathway were strongly changed in cells after KW2449 treatment, indicating a potential role of the pathway in drug resistance. We found that KW2449 caused a dose-dependent, biphasic induction of AKT phosphorylation at Ser473 in mutant-PTEN cells. Co-treatment with the inhibitors of its upstream signaling completely abolished the reactivation of AKT phosphorylation by KW2449 and reversed the drug resistant phenotype. These data suggest that reactivation of AKT phosphorylation at Ser473 is a key factor to confer drug resistant phenotype of mutant-PTEN cells to the dual inhibitors and that proper drug combinations that shut down AKT reactivation is necessary for the effective treatment of mutant-PTEN cancer with the dual inhibitors in clinical settings.

Introduction

Phosphatase and tensin homolog (PTEN) gene is a well-known tumor suppressor, which locates on chromosome 10, with dual functional phosphatase domain and C2 domain [1,2]. PTEN regulates apoptosis and control cell proliferation by negatively regulating PI3K/AKT pathway [2]. It also plays critical roles in chromosome stability and DNA double stand break (DSB) repair pathway [3,4]. Loss-of-function mutations or deletions in PTEN are found in many cancers. Mutations of PTEN in germ line cells can cause PTEN hamartoma tumor syndromes (PHTS), such as Cowden syndrome [5]. The loss of heterozygosity (LOH) of PTEN and loss of expression by epigenetic silencing have been found in colorectal, breast, prostate cancer and other cancers, making it as a key tumor suppressor in wide variety of tumor types [[6], [7], [8], [9], [10]]. Mutated or deleted PTEN is reported highly linked to drug resistance in leukemia, breast cancer, prostate cancer, glioblastoma and many other cancers [[11], [12], [13], [14], [15], [16]]. In 2010, PTEN and RAS abnormalities were reported to show negative impact to the therapy outcomes of pediatric patients with T-cell ALL through PTEN/NOTCH1/FBXW7 and RAS/RAF/MEK/ERK pathways respectively [11]. PTEN mutant lacking both lipid and protein phosphatase activity conferred more resistance to doxorubicin in MCF-7 breast cancers [12]. An RNA interfering (RNAi) screening showed that PTEN was the only modulator and activated PI3K pathway served as a marker in trastuzumab resistant breast cancer [13]. Singly targeting PI3K or mTOR by the inhibitors also did not cause significant cell death in PTEN mutated gliomas, indicating the drug resistance resulting from PTEN deficiency [14]. Two p110β inhibitors, GSK2636771 and AZD6482 showed drug resistance in PTEN mutant endometrioid endometrial cancers (EECs) [15]. Ovarian cancer cells with low PTEN expression showed drug resistance to cisplatin due to the activation of AKT, followed by the inhibition of Bax translocation to mitochondria [16]. As drug resistance is a major cause of tumor recurrence, metastasis and poor prognosis [17], measuring drug response in cancer cell lines and identifying mechanisms of drug resistance are essential for effective cancer treatment [18].

Colorectal cancer (CRC) is the second leading cause of cancer death in the US and the drug resistance limits the effectiveness and prognosis of anticancer therapies for CRC [19]. As PTEN is often found mutated or deleted in CRC [[6], [7], [8]], synthetic lethality approach targeting mutant-PTEN (mtPTEN) cancer may provide new treatment opportunities for such cancer types [20]. Based on this notion, we initially aimed to identify drugs that are selective for mtPTEN CRC cells using a synthetic lethality epigenetics drug screening with PTEN-isogenic CRC pair. From the screening we failed to identify synthetic lethality compounds for PTEN mutation, but unexpectedly found that mtPTEN CRC cells were strongly resistant to the dual inhibitors of fms like tyrosine kinase 3 (FLT3) and aurora kinase A (AURKA). Dual inhibitors of FLT3 and AURKA, including KW2449 and ENMD-2076, are anticancer agents that target multikinases with particular potency and specificity on FLT3 and AURKA [21,22]. They are being actively investigated in Phase I and II clinical trials for the treatment of various types of cancer (https://clinicaltrials.gov/). Although FLT3 has been a main target for treating acute myeloid leukemia (AML), recent studies suggested that FLT3 amplification could also contribute to the poor prognosis of metastatic CRC [23]. According to The Cancer Genome Atlas (TCGA) data analyses, the incidence of FLT3 amplification was highest in CRC (34.6%) in the frequency among all FLT3-amplified cancer [24]. On the other hand, AURKA is also often over-expressed in CRC, making it as an important CRC drug target [25]. These studies call for possible use of the dual inhibitors of FLT3/AURKA for treating CRC and other solid cancers [26].

The present study shows that PTEN status can be an important factor determining CRC cell sensitivity to the dual inhibitors of FLT3/AURKA. We further elaborate a plausible mechanism of the mtPTEN-driven drug resistance in CRC and provide effective drug combinations to restore drug sensitivity of mtPTEN CRC cells to the dual inhibitors.

Section snippets

Cell culture and reagents

HCT116 cells and PC3 cells were purchased from ATCC (American Type Tissue Collection, Manassas, VA, USA) and cultured in RPMI-1640 with 10% Fetal Bovine Serum (#26140079, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% penicillin and streptomycin (#15140163, Thermo Fisher Scientific) at 37° of CO2 incubator. The epigenetics drug library, KW2449, ENMD-2076, Aurora kinase inhibitor I, LY294002, Wortmannin, MK2206, Torin, Rapamycin, PD98059 and BX795 were purchased from Selleck

Synthetic lethality screening identified that mtPTEN CRC cells are resistant to KW2449 and ENMD-2076

To perform the synthetic lethality drug screening for PTEN mutation, we generated mtPTEN HCT116 CRC cell line by knocking out (KO) PTEN gene with CRISPR-Cas9 gene editing system. HCT116 cells expressing wtPTEN was transfected with CRISPR/Cas-9 plasmid, sgRNAs targeting human PTEN gene and homology-directed repair (HDR) donor plasmid containing RFP and puromycin selection markers (Supplementary Fig. S1A). PTEN KO (mtPTEN) clones were selected with RFP fluorescence and puromycin resistance (

Discussion

Mutated or deleted PTEN is reported highly linked to tumor recurrence due to its chemo-resistance effects [[35], [36], [37], [38]]. PTEN loss drives cancer cells to confer resistant not only to chemotherapeutic agents, but also to a number of hormonal and targeted therapy agents. It drives estrogen receptor (ER)-positive breast cancer cells resistance to hormonal therapy drugs tamoxifen and fulvestrant [37,39], non-small cell lung cancer cells resistance to epidermal growth factor receptor

Acknowledgements

We thank to the members of the Genomics and Bioinformatics Core of FHS and FHS Animal Facility at the University of Macau for experimental and technical supports. Transcriptome profiling work was performed in part at the High Performance Computing Cluster (HPCC) which is supported by Information and Communication Technology Office (ICTO) of the University of Macau. This work was supported by the Multi-Year Research Grant of the University of Macau (MYRG2015-00181-FHS and MYRG2017-00176-FHS to

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