Luis E. Arias-Romero

A tale of two Paks

Paks (p21‐activated kinases) are effectors for the small GTPases Cdc42 (cell division cycle 42) and Rac that play fundamental roles in a wide range of cellular processes, including cell morphology, motility, survival, gene transcription, apoptosis and hormone signalling. These enzymes are widely expressed in numerous tissues and are activated by extracellular signals through GTPase‐dependent and ‐independent mechanisms. On the basis of structural and biochemical features, the Pak family members have been classified into two groups, comprising three members each. The two Pak groups have generally been considered as two halves of a single entity, but accumulating evidence indicates that this is not so. In this review, we discuss recent advances in our understanding of the structure, regulation and function of these kinases, emphasizing the many differences between these two groups of signalling proteins.

A Rac-Pak signaling pathway is essential for ErbB2-mediated transformation of human breast epithelial cancer cells.

The activation of receptor tyrosine kinases, particularly ErbB2, has an important role in the genesis of breast cancer. ErbB2 kinase activity promotes Ras-mediated stimulation of downstream protein kinase cascades, including the Ras/Raf-1/MAPK/ERK kinase (Mek)/extracellular signal-regulated kinase (Erk) pathway, leading to tumor cell growth and migration. Signaling through the Ras–Erk pathway can be influenced by p21-activated kinase-1 (Pak1), an effector of the Rho family GTPases Rac and Cdc42. In this study, we asked if ErbB2 expression correlates with Pak1 and Erk activity in human breast cancer specimens, and if Pak1 signaling is required for ErbB2 transformation in a three-dimensional (3D) in vitro setting and in xenografts. We found a correlation between ErbB2 expression and activation of Pak in estrogen receptor-positive human breast tumor samples and observed that in 3D cultures, activation of Rac–Pak1 pathway by ErbB2 homodimers induced growth factor-independent proliferation and promoted disruption of 3D mammary acinar-like structures through activation of the Erk and Akt pathways. Further, we found that inhibition of Pak1 by small molecules compromised activation of Erk and Akt, resulting in reversion of the malignant phenotype and restoration of normal acinar architecture. Finally, ErbB2-amplified breast cancer cells expressing a specific Pak inhibitor showed delayed tumor formation and downregulation of Erk and Akt signaling in vivo. These data imply that the Rac–Pak pathway is vital to ErbB2-mediated transformation and that Pak inhibitors represent plausible drug targets in breast cancers in which ErbB2 signaling is activated.

Activation of Src by Protein Tyrosine Phosphatase 1B Is Required for ErbB2 Transformation of Human Breast Epithelial Cells

Protein tyrosine phosphatase (PTP) 1B plays a major role in inhibiting signaling from the insulin and leptin receptors. Recently, PTP1B was found to have an unexpected positive role in ErbB2 signaling in a mouse model of breast cancer, but the mechanism underlying this effect has been unclear. Using human breast epithelial cells grown in a three-dimensional matrix, we found that PTP1B, but not the closely related enzyme T-cell PTP, is required for ErbB2 transformation in vitro. Activation of ErbB2, but not ErbB1, increases PTP1B expression, and increased expression of PTP1B activates Src and induces a Src-dependent transformed phenotype. These findings identify a molecular mechanism by which PTP1B links an important oncogenic receptor tyrosine kinase to signaling pathways that promote aberrant cell division and survival in human breast epithelial cells.

Interaction with LC8 is required for Pak1 nuclear import and is indispensable for zebrafish development.

Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.

Pak1 Kinase Links ErbB2 to β-Catenin in Transformation of Breast Epithelial Cells

p21-Activated kinase-1 (Pak1) is frequently upregulated in human breast cancer and is required for transformation of mammary epithelial cells by ErbB2. Here, we show that loss of Pak1, but not the closely related Pak2, leads to diminished expression of β-catenin and its target genes. In MMTV-ErbB2 transgenic mice, loss of Pak1 prolonged survival, and mammary tissues of such mice showed loss of β-catenin. Expression of a β-catenin mutant bearing a phospho-mimetic mutation at Ser 675, a specific Pak1 phosphorylation site, restored transformation to ErbB2-positive, Pak1-deficient mammary epithelial cells. Mice bearing xenografts of ErbB2-positive breast cancer cells showed tumor regression when treated with small-molecule inhibitors of Pak or β-catenin, and combined inhibition by both agents was synergistic. These data delineate a signaling pathway from ErbB2 to Pak to β-catenin that is required for efficient transformation of mammary epithelial cells, and suggest new therapeutic strategies in ErbB2-positive breast cancer.

Targeting Cdc42 in cancer.

Introduction: The Rho GTPases are a family of proteins that control fundamental cellular processes in response to extracellular stimuli and internal programs. Rho GTPases function as molecular switches in which the GTP-bound proteins are active and GDP-bound proteins are inactive. This article will focus on one Rho family member, Cdc42, which is overexpressed in a number of human cancers, and which might provide new therapeutic targets in malignancies. Areas covered: In this article, the key regulators and effectors of Cdc42 and their molecular alterations are described. The complex interactions between the signaling cascades regulated by Cdc42 are also analyzed. Expert opinion: While mutations in Cdc42 have not been reported in human cancer, aberrant expression of Cdc42 has been reported in a variety of tumor types and in some instances has been correlated with poor prognosis. Recently, it has been shown that Cdc42 activation by oncogenic Ras is crucial for Ras-mediated tumorigenesis, suggesting that targeting Cdc42 or its effectors might be useful in tumors harboring activating Ras mutations.

p21-activated kinases in Erbb2-positive breast cancer: A new therapeutic target?

The activation of receptor tyrosine kinases, particularly ErbB2, has been linked to the genesis and progression of breast cancer. Two of the central signaling pathways activated by ErbB2 are the Ras/Raf-1/Mek/Erk pathway, which plays an important role in tumor cell growth and migration, and the PI3K/Akt pathway, which plays an important role in cell survival. Recently, we and others have shown that signaling through the Ras-Erk pathway can be influenced by p21-activated kinase 1 (Pak1), an effector of the Rho family GTPases Rac and Cdc42. Expression of activated forms of Rac promotes activation of Erk through mechanisms involving Pak1 phosphorylation of Raf-1 and Mek1. In addition, Pak1 has also been implicated in the activation of Akt. However, our understanding regarding the degree to which Rho GTPases, and their effectors such as Pak1, contribute to ErbB2-mediated signaling is very limited.

H-ras Inhibits the Hippo Pathway by Promoting Mst1/Mst2 Heterodimerization

The protein kinases Mst1 and Mst2 have tumor suppressor activity, but their mode of regulation is not well established. Mst1 and Mst2 are broadly expressed and may have certain overlapping functions in mammals, as deletions of both Mst1 and Mst2 together are required for tumorigenesis in mouse models [1-3]. These kinases act via a three-component signaling cascade comprising Mst1 and Mst2, the protein kinases Lats1 and Lats2, and the transcriptional coactivators Yap and Taz [4-6]. Mst1 and Mst2 contain C-terminal SARAH domains that mediate their homodimerization as well as heterodimerization with other SARAH domain-containing proteins, which may regulate Mst1/Mst2 activity. Here we show that, in addition to forming homodimers, Mst1 and Mst2 heterodimerize in cells, this interaction is mediated by their SARAH domains and is favored over homodimers, and these heterodimers have much-reduced protein kinase activity compared to Mst1 or Mst2 homodimers. Mst1/Mst2 heterodimerization is strongly promoted by oncogenic H-ras, and this effect requires activation of the Erk pathway. Cells lacking Mst1, in which Mst1/Mst2 heterodimers are not possible, are resistant to H-ras-mediated transformation and maintain active hippo pathway signaling compared to wild-type cells or cells lacking both Mst1 and Mst2. Our results suggest that H-ras, via an Erk-dependent mechanism, downregulates Mst1/Mst2 activity by inducing the formation of inactive Mst1/Mst2 heterodimers.

Lack of STAT6 Attenuates Inflammation and Drives Protection against Early Steps of Colitis-Associated Colon Cancer

STAT6 plays a role in inflammation and in some malignancies. It was found to fuel colitis-related colorectal cancer in a mouse model. Its absence decreased the number of tumors by inhibiting early steps in the progression to colon cancer. Colitis-associated colon cancer (CAC) is one of the most common malignant neoplasms and a leading cause of death. The immunologic factors associated with CAC development are not completely understood. Signal transducer and activator of transcription 6 (STAT6) is part of an important signaling pathway for modulating intestinal immune function and homeostasis. However, the role of STAT6 in colon cancer progression is unclear. Following CAC induction in wild-type (WT) and STAT6-deficient mice (STAT6–/–), we found that 70% of STAT6–/– mice were tumor-free after 8 weeks, whereas 100% of WT mice developed tumors. STAT6–/– mice displayed fewer and smaller colorectal tumors than WT mice; this reduced tumorigenicity was associated with decreased proliferation and increased apoptosis in the colonic mucosa in the early steps of tumor progression. STAT6–/– mice also exhibited reduced inflammation, diminished concentrations COX2 and nuclear β-catenin protein in the colon, and decreased mRNA expression of IL17A and TNFα, but increased IL10 expression when compared with WT mice. Impaired mucosal expression of CCL9, CCL25, and CXCR2 was also observed. In addition, the number of circulating CD11b+Ly6ChiCCR2+ monocytes and CD11b+Ly6ClowLy6G+ granulocytes was both decreased in a STAT6-dependent manner. Finally, WT mice receiving a STAT6 inhibitor in vivo confirmed a significant reduction in tumor load as well as less intense signs of CAC. Our results demonstrate that STAT6 is critical in the early steps of CAC development for modulating inflammatory responses and controlling cell recruitment and proliferation. Thus, STAT6 may represent a promising target for CAC treatment. Cancer Immunol Res; 5(5); 385–96. ©2017 AACR.

p21 Activated kinase 1: Nuclear activity and its role during DNA damage repair

p21-activated kinase 1 (PAK1) is a serine/threonine kinase activated by the small GTPases Rac1 and Cdc42. It is located in the chromosome 11q13 and is amplified and/or overexpressed in several human cancer types including 25-30% of breast tumors. This enzyme plays a pivotal role in the control of a number of fundamental cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, it is well documented that PAK1 also plays crucial roles in the nucleus participating in mitotic events and gene expression through its association and/or phosphorylation of several transcription factors, transcriptional co-regulators and cell cycle-related proteins, including Aurora kinase A (AURKA), polo-like kinase 1 (PLK1), the forkhead transcription factor (FKHR), estrogen receptor α (ERα), and Snail. More recently, PAK signaling has emerged as a component of the DNA damage response (DDR) as PAK1 activity influences the cellular sensitivity to ionizing radiation and promotes the expression of several genes involved in the Fanconi Anemia/BRCA pathway. This review will focus on the nuclear functions of PAK1 and its role in the regulation of DNA damage repair.