Small Molecule Inhibition of Group I p21-Activated Kinases in Breast Cancer Induces Apoptosis and Potentiates the Activity of Microtubule Stabilizing Agents

Christy C Ong; Sarah Gierke; Cameron Pitt; Meredith Sagolla; Christine K Cheng; Wei Zhou; Adrian M Jubb; Laura Strickland; Maike Schmidt; Sergio G Duron; David A Campbell; Wei Zheng; Seameen Dehdashti; Min Shen; Nora Yang; Mark L Behnke; Wenwei Huang; John C McKew; Jonathan Chernoff; William F Forrest; Peter M Haverty; Suet-Feung Chin; Emad A Rakha; Andrew R Green; Ian O Ellis; Carlos Caldas; Thomas O'Brien; Lori S Friedman; Hartmut Koeppen; Joachim Rudolph; Klaus P Hoeflich

Disclosures

Breast Cancer Res. 2015;17(59) 

In This Article

Abstract and Introduction

Abstract

Introduction: Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis.

Methods: PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n = 980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting.

Results: We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P = 1.29 × 10−4 and P = 0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis.

Conclusions: Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.

Introduction

The p21-activated kinases (PAKs) have generated significant interest as therapeutic targets in cancer.[1,2] The PAK family is comprised of six members and is subdivided into two groups (Groups I and II) based on sequence and structural homology. PAKs are currently amongst the most well-characterized effector proteins of the Ras-related C3 botulinum toxin substrate 1 (Rac) and cell division control protein 42 (Cdc42). These GTPases stimulate PAK catalytic activity by relieving an intramolecular interaction between the kinase and autoinhibitory domains. The kinase domains of Group I versus II PAKs share approximately 50% identity and also share homology with additional members of the sterile-20 (STE20) subfamily of the kinome that are upstream activators of mammalian mitogen-activated protein kinase (MAPK) pathways.

PAK1 signaling has been shown to be important for regulating cytoskeletal organization and cell migration via both its catalytic activity and protein-protein interactions. For instance, PAK1 modulates the activity of myosin II (an actin interacting motor protein that can drive cell contractility), LIM-kinase (involved in actin polymerization through inactivation of cofilin family proteins) and filamin A (a large actin-binding protein that induces membrane ruffling).[3] PAK1 is also involved in the phosphorylation of proteins that control microtubule dynamics such as stathmin, which destabilizes microtubules by binding tubulin dimers to inhibit tubulin polymerization and promote microtubule disassembly.[4] In addition, PAK1 phosphorylates tubulin cofactor B to augment heterodimerization of tubulin[5] as well as dynein light chain 1 which is a component of the cytoplasmic dynein complex that moves along with microtubules.[6] To date, the evidence for the role of PAK1 in microtubule remodeling comes primarily from overexpression and genetic studies. For instance, PAK1−/− mouse embryonic fibroblasts display decreased microtubule regrowth and polymerization compared with wild-type cells, and the reciprocal phenotypic was observed using MCF7 breast cancer cells overexpressing PAK1.[7] The contribution of PAK1 catalytic activity to microtubule dynamics has yet to be thoroughly explored.

In addition to its role in regulation of the cytoskeleton, PAK1 has been implicated in cellular processes that directly contribute to tumorigenesis, including growth factor pathways, cell proliferation, and pro-survival signaling.[8] PAK1 is also an effector of well-established oncogenes, such as the Ras small monomeric GTPase which is mutated in approximately 30% of human tumors. Given that Rac and Cdc42 lie downstream of Ras,[9,10] several groups have evaluated the contribution of PAKs to Ras-driven cellular transformation and in vivo tumorigenesis.[11,12] For instance, PAK1 deletion in a mouse model of Ras-driven cutaneous squamous cell carcinoma led to markedly decreased tumorigenesis and progression, which was accompanied by attenuated signaling through MAPK and cytoskeletal pathways.[11]

In terms of direct dysregulation in cancer, PAK1 is amplified, overexpressed or hyperactivated in several tumor subtypes.[1,13] Of note, focal genomic amplification of PAK1 at 11q14.1 has been reported for hormone receptor-positive breast carcinoma.[14,15] Analysis of breast cancer cell lines with PAK1 genomic copy number gain using RNA interference approaches revealed dependence on PAK1 expression for cell survival[14] and transformation.[16] Consistent with these findings, functional studies using transgenic mouse models have also demonstrated that overexpression of PAK1 in the mammary gland promotes the formation of preneoplastic lesions and breast tumors[17] and that PAK1 contributes to human endothelial growth factor receptor 2 (HER2)/Neu-driven tumorigenesis.[18]

However, given this emerging body of work, a detailed assessment of PAK1 copy number alteration and validation experiments using small molecule inhibitors to evaluate PAK1 catalytic inhibition in breast cancer are still lacking. Moreover, the potential efficacy of PAK1 inhibition in combination with additional inhibitors of cytoskeletal organization has yet to be examined. Herein, we demonstrate that PAK1 gene amplification and protein overexpression are associated with poor clinical outcome in a large collection of luminal breast cancers. We also introduce a novel ATP-competitive small molecule inhibitor of group I PAKs, FRAX1036, and demonstrate sensitivity of PAK1-amplified breast cancer cells to this compound. Taken together, these results suggest that further investigation of PAK1 as a therapeutic target in breast cancer is warranted. Given that PAK1 regulates the cytoskeleton and microtubule inhibitors are used as standard-of-care chemotherapy in advanced breast cancer, we explored the molecular and cellular mechanisms for this therapeutic combination and showed increased anti-tumor efficacy in breast cancer cells.

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