1. Bruton’s tyrosine kinase is a potential therapeutic target in solid tumor cancers
Leila Kokabee*, Xianhui Wang, Cheryl Eifert, Douglas S. Conklin
Cancer Research Center and Department of Biomedical Sciences; State University of New York; University at Albany; Rensselaer, NY, USA
Abstract
Therapeutic resistance and metastasis continue to be major challenges in the clinical management of solid tumor cancers such as breast and prostate cancers. The identification of new targets with roles in cancer cell survival is critically important. Tyrosine kinases orchestrate key cellular signaling pathways and their dysregulation is often associated with cellular transformation and cancer. Therefore, we have performed a large-scale RNA interference (RNAi) screen for tyrosine kinase genes whose function is critical for breast cancer cell survival. Bruton‘s tyrosine kinase (BTK), which has been studied in B cell development, was among those genes whose knockdown causes the most significant reduction in survival. We find that BTK is expressed in solid tumor cancer cells, with the alternative BTK-C isoform predominantly expressed in breast and prostate cancer cells and tumors. Down regulation of this BTK with RNAi (shRNA, siRNA) or inhibition with BTK-specific inhibitors such as Ibrutinib, AVL-292 decrease cell survival and induce apoptosis in breast and prostate cancer cells. Microarray results show that inhibiting BTK increases apoptosis related genes, while overexpression of BTK-C is associated with elevated expression of genes with function related to cell adhesion, cytoskeletal structure and the extracellular matrix. In addition, our in vivo studies shows, Ibrutinib inhibits HER2+ xenograft tumor growth. Also, we find, inhibition of BTK prevents activation of the AKT signaling pathway by NRG or EGF that has been shown to promote growth factor-driven lapatinib resistance in HER2+ breast cancer cells. Our work has shown BTK-C is a survival factor for solid tumor cancer cells and it represents both a potential biomarker and novel therapeutic target for solid tumor cancers, especially in breast and prostate cancers and inhibiting BTK can prevent therapeutic escape in solid tumor cancer cells.
Cell survival
Apoptosis
(Caspase 3) A B C
whose knockdown causes the most significant reduction in BT474 cellular proliferation (Fig.1B). Surprisingly, the second tyrosine kinase with the most significant reduction was Bruton’s tyrosine kinase (BTK), a non-receptor tyrosine kinase which has an important role in B cell development but normally is not expected to be present in breast cancer.
Figure 3. Targeting BTK with siRNA or inhibition with BTK inhibitors decrease cell survival and induce apoptosis in solid tumor cancer cells (A) Knock down with siRNAs specific for BTK-C decrease cell survival in solid tumor cancer cells (B) and leads to widespread cell death and apoptosis. (C) Inhibition with BTK inhibitors such as Ibrutinib, AVL-292 decrease cell survival in solid tumor cancer cells. Transfected or treated cells were counted and the 96h to 24h or 72h to 24hratio was calculated and expressed as % of the control. Mean of triplicate assays ± S.D. Student t-test, *p < 0.05 A B
Figure 1. An RNAi screen targets tyrosine kinase genes in a HER2 positive breast cancer cell. (A) BT474 cells were transfected with 234 shRNAs constructs to target 83 tyrosine kinase genes. The 25 of 83 TK genes showed more than 50% reduction in BT474 cellular proliferation. (B) Some of tyrosine kinases with most significant reduction in BT474 cell proliferation.
BT474 breast cancer cells express a novel variant of the BTK message from an alternative promoter
Our results indicate that expression of the BTK-C transcript in BT474 breast cancer cells is driven by an alternate promoter located upstream from the published BTK-A promoter. The first exons from the BTK-A and BTK-C isoforms utilize different donor sites to splice into a common acceptor site, located within exon 2, to yield the mature BTK-A and BTK-C mRNA isoforms (Fig. 2A). Due to the additional sequence, the BTK-C message encodes a product that contains an amino-terminal 34 amino acid extension to the BTK-A protein (Fig. 2B,C). a c
Introduction
Therapeutic resistance and metastasis continue to be major challenges in the clinical management of solid tumor cancers such as breast and prostate cancers. Identifying novel mediators that regulate the growth and survival of cancer cells has accelerated the development of therapies that have steadily improved clinical outcomes in cancer patients. Protein tyrosine kinase inhibitors are among the most promising targeted therapies. Dysregulation of these enzymes causes increases in tumor cell proliferation and abrogation of apoptotic pathways while promoting angiogenesis and metastasis. We performed a genome-wide RNAi screen for tyrosine kinase genes whose function is critical for breast cancer cell survival. Bruton’s Tyrosine kinase (BTK) was among those genes whose knockdown causes the most significant reduction in survival. BTK is a critical regulator of B cell receptor (BCR) signaling. Mutations in the btk gene lead to B cell deficiency manifested as X-linked agammaglobulinemia in humans. In haematopoietic cells, BTK is involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B lymphocytes. Given the importance of BTK activity in solid tumor cancer cells, these data indicate that, in addition to its utility in haematopoietic malignancies, targeting BTK may be a potent therapeutic approach for solid tumor cancers.
Figure 4. BTK-C alters gene expression in solid tumor cancer cells. RNA from Ibrutinib treated cells (A) or cells overexpressing BTK-A and BTK-C or empty vector (B) were used for microarray experiments. Results of the Functional Annotation Clustering Analysis after GO-term enrichment of up-regulated and down-regulated genes using the DAVID Bioinformatics Resource. BP; Biological Process, CC; Cellular Component and MF; Molecular Function. Venn diagram showing the number of genes those are up-regulated or down-regulated with overexpression of BTK-A or BTK-C in solid tumor cancer cells. A
Ibrutinib inhibits HER2+ xenograft tumor growth.
Results from molecular experiments carried out in vitro, showed inhibiting BTK might be useful in inhibiting tumor progression. We assessed the effect of ibrutinib on xenografts of SKBR3 in NOD/SCID mice. Our results indicate Ibrutinib treatment inhibits tumor growth when administered to animals. B
( B-cells )
(Solid tumor)
+34 AA A B C
Prostate cancer cell lines
Breast cancer cell lines
Figure 2. An alternative form of the BTK transcript is present in breast cancer cells. (A) Schematic representation of alternative splicing from alternative first exons of both isoforms, sequence is identical from exon 2 through exon 19. (B) Due to the additional sequence, the BTK-C message encodes a product that contains an amino-terminal 34 amino acid extension to the BTK-A protein. (C) The BTK-C, an 80 kD product, total lysate from solid tumors and Namalwa B-cells subjected to blotted and probed with an anti-BTK antibody
Figure 5. Ibrutinib inhibits Her2+ breast cancer cell growth in vivo. (A) Images of tumors formed in animals after mammary fat pad injection of SKBR3 cancer cells. Animal were treated with vehicle, lapatinib and ibrutinib for four week (B) tumor growth curves obtained following fat pad injection of SKBR3 cells. The data represent the mean SE (n=5,*P<0.01).
Targeting BTK with siRNA or inhibition with BTK inhibitors decrease cell survival and induce apoptosis in solid tumor cancer cells
The results of BTK-C specific siRNAs knockdown experiments established that these siRNAs silence the BTK-C isoform transcript, specifically and leads to a statistically significant decrease in solid tumor cancer cell number (Fig.3A) and induce apoptosis (Fig.3B) in these solid tumor cancer cells. Additionally, inhibiting BTK with BTK inhibitors such as Ibrutinib, AVL-292 decrease cell survival in solid tumor cancer cells (Fig.3C).
Results
BTK identified as a survival factor in an RNAi knockdown screen of the TKs in BT474 breast cancer cells
To identify additional survival kinases and pathways in breast cancer, we have performed a large-scale RNA interference (RNAi) screen of the protein tyrosine kinases in the BT474, HER2 positive breast cancer cell line. RNAi was used to knock down the human genome’s tyrosine kinases to evaluate the functional contribution of each tyrosine kinase to BT474 breast cancer cell viability. The 236 short-hairpin RNAs (shRNAs) from the pSHAG-MAGIC2 (pSM2) library targeted 82 of the 90 human tyrosine kinase genes. About 30% (25 of the 82 genes) of TK genes were inhibited using shRNAs led to a 50% decrease in BT474 cellular proliferation compared to the control (Fig.1A). ABL2, FES, NTRK2, PTK2B, PTK9, EGFR2, EPHA1, LYN and BTK were among the kinases
Conclusion
Novel isoform of BTK predominantly expressed in breast and prostate cancer cells and tumors.
BTK-C is a survival factor for solid tumor cancer cells.
Microarray results along with data from migration and invasion assay suggest that BTK-C expression in solid tumor cancer cells might be involved in metastasis signaling
In vivo studies shows, Ibrutinib inhibits HER2+ xenograft tumor growth.
These results suggest targeting BTK may improve the management of treating breast and prostate cancer patients
BTK-C alters gene expression in solid tumor cancer cells
Microarray results show inhibition with a BTK inhibitor increases expression of apoptosis related genes (Fig.4A). Overexpression of BTK-C is associated with elevated expression of genes related to cell adhesion, cytoskeletal structure and extracellular matrix (Fig.4B).