Suresh K. Rayala

PAK Signaling in Oncogenesis

The p21-activated kinase (PAK) family of serine/threonine kinases is important in physiological processes including motility, survival, mitosis, transcription and translation. PAKs are evolutionally conserved and widely expressed in a variety of tissues and are often overexpressed in multiple cancer types. Depending on structural and functional similarities, the six members of PAK family are divided into two groups with three members in each group. Group I PAKs are activated by extracellular signals through GTPase-dependent and GTPase-independent mechanisms. In contrast, group II PAKs are constitutively active. Over the years, accumulating data from tissue culture models and human tumors has increased our understanding about the biology of PAK family members. In this review, we have summarized the complex regulation of PAK and its downstream diverse myriads of effectors, which in turn are responsible for the biological effects of PAK family of kinases in cancer cells.

Nanomedicine: towards development of patient-friendly drug-delivery systems for oncological applications.

The focus on nanotechnology in cancer treatment and diagnosis has intensified due to the serious side effects caused by anticancer agents as a result of their cytotoxic actions on normal cells. This nonspecific action of chemotherapy has awakened a need for formulations capable of definitive targeting with enhanced tumor-killing. Nanooncology, the application of nanobiotechnology to the management of cancer, is currently the most important area of nanomedicine. Currently several nanomaterial-based drug-delivery systems are in vogue and several others are in various stages of development. Tumor-targeted drug-delivery systems are envisioned as magic bullets for cancer therapy and several groups are working globally for development of robust systems.

An inherent role of microtubule network in the action of nuclear receptor

Estrogen receptor α (ERα) functions as both a transcription factor and a mediator of rapid estrogen signaling. Recent studies have shown a role for ERα-interacting membranous and cytosolic proteins in ERα action, but our understanding of the role of the microtubule network in the modulation of ERα signaling remains unclear. Here we found that endogenous ERα associates with microtubules through the microtubule-binding protein hematopoietic PBX-interaction protein (HPIP). Biochemical and RNA-interference studies demonstrated that HPIP influences ERα-dependent rapid estrogen signaling by acting as a scaffold protein and recruits Src kinase and the p85 subunit of phosphatidylinositol 3-kinase to a complex with ERα, which in turn stimulates AKT and MAPK. We also found that ERα interacts with β-tubulin through HPIP. Destabilization of microtubules activated ERα signaling, whereas stabilization of microtubules repressed ERα transcriptional activity in a HPIP-dependent manner. These findings revealed a role for HPIP–microtubule complex in regulating 17β-estradiol–ERα responses in mammalian cells and discovered an inherent role of microtubules in the action of nuclear receptor.

MTA1 promotes STAT3 transcription and pulmonary metastasis in breast cancer

Overexpression of the prometastatic chromatin modifier protein metastasis tumor antigen 1 (MTA1) in human cancer contributes to tumor aggressiveness, but the role of endogenous MTA1 in cancer has not been explored. Here, we report the effects of selective genetic depletion of MTA1 in a physiologically relevant spontaneous mouse model of breast cancer pulmonary metastasis. We found that MTA1 acts as a mandatory modifier of breast-to-lung metastasis without effects on primary tumor formation. The underlying mechanism involved MTA1-dependent stimulation of STAT3 transcription through action on the MTA1/STAT3/Pol II coactivator complex, and, in turn, on the expression and functions of STAT3 target genes including Twist1. Accordingly, we documented a positive correlation between levels of MTA1 and STAT3 in publicly available breast cancer data sets. Together, our findings reveal an essential modifying role of the physiologic level of MTA1 in supporting pulmonary metastasis of breast cancer.

Serine 88 Phosphorylation of the 8-kDa Dynein Light Chain 1 Is a Molecular Switch for Its Dimerization Status and Functions

Dynein light chain 1 (DLC1, also known as DYNLL1, LC8, and PIN), a ubiquitously expressed and highly conserved protein, participates in a variety of essential intracellular events. Transition of DLC1 between dimer and monomer forms might play a crucial role in its function. However, the molecular mechanism(s) that control the transition remain unknown. DLC1 phosphorylation on Ser88 by p21-activated kinase 1 (Pak1), a signaling nodule, promotes mammalian cell survival by regulating its interaction with Bim and the stability of Bim. Here we discovered that phosphorylation of Ser88, which juxtapose each other at the interface of the DLC dimer, disrupts DLC1 dimer formation and consequently impairs its interaction with Bim. Overexpression of a Ser88 phosphorylation-inactive DLC1 mutant in mammary epithelium cells and in a transgenic animal model caused apoptosis and accelerated mammary gland involution, respectively, with increased Bim levels. Structural and biophysical studies suggested that phosphorylation-mimicking mutation leads to dissociation of the DLC1 dimer to a pure folded monomer. The phosphorylation-induced DLC1 monomer is incapable of binding to its substrate Bim. These findings reveal a previously unrecognized regulatory mechanism of DLC1 in which the Ser88 phosphorylation acts as a molecular switch for the transition of DLC1 from dimer to monomer, thereby modulating its interaction with substrates and consequently regulating the functions of DLC1.

Phosphorylation-dependent Regulation of Stability and Transforming Potential of ETS Transcriptional Factor ESE-1 by p21-activated Kinase 1

Differential phosphorylation of transcription factors by signal transduction pathways play an important role in regulation of gene expression and functions. ESE-1 is an epithelium-specific ETS transcription factor that transforms human breast epithelial cells through a serine- and aspartic acid-rich domain (SAR) by an unknown cytoplasmic mechanism. Here we found that a signaling kinase, p21-activated kinase-1 (Pak1), interacts with and phosphorylates ESE-1. Interestingly, Pak1 selectively phosphorylates ESE-1 at Ser207, which is located within the SAR domain. A S207A substitution in ESE-1 reduced its ability to transform breast cancer cells. We also found that ESE-1 is a labile protein and by interacting with F-box-binding protein β-TrCP, undergoes ubiquitin-dependent proteolysis. Intriguingly, Pak1 phosphorylation inactive mutant ESE1-S207A is more unstable than either wild-type ESE-1 or its Pak1 phosphorylation mimetic mutant, i.e. ESE1-S207E. These findings provide novel insights into the mechanism of transformation potential of ESE-1 and discovered that ESE-1 functions are coordinately regulated by Pak1 phosphorylation and β-TrCP-dependent ubiquitin-proteasome pathways.

Transcriptional regulation of fibronectin by p21-activated kinase-1 modulates pancreatic tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) is the eighth largest cause of cancer-related mortality across the world, with a median 5-year survival rate of less than 3.5%. This is partly because the molecules and the molecular mechanisms that contribute to PDAC are not well understood. Our goal is to understand the role of p21-activated kinase 1 (Pak1) signaling axis in the progression of PDAC. Pak1, a serine/threonine kinase, is a well-known regulator of cytoskeletal remodeling, cell motility, cell proliferation and cell survival. Recent reports suggest that Pak1 by itself can have an oncogenic role in a wide variety of cancers. In this study, we analyzed the expression of Pak1 in human pancreatic cancer tissues and found that Pak1 levels are significantly upregulated in PDAC samples as compared with adjacent normals. Further, to study the functional role of Pak1 in pancreatic cancer model systems, we developed stable overexpression and lentiviral short hairpin RNA-mediated knockdown (KD) clones of Pak1 and studied the changes in transforming properties of the cells. We also observed that Pak1 KD clones failed to form tumors in nude mice. By adopting a quantitative PCR array-based approach, we identified fibronectin, a component of the extracellular matrix and a mesenchymal marker, as a transcriptional target of Pak1 signaling. The underlying molecular mechanism of Pak1-mediated transformation includes its nuclear import and recruitment to the fibronectin promoter via interaction with nuclear factor-κB (NF-κB)–p65 complex. To our knowledge, this is the first study illustrating Pak1–NF-κB–p65-mediated fibronectin regulation as a potent tumor-promoting mechanism in KRAS intact model.

Tumor targeting using polyamidoamine dendrimer-cisplatin nanoparticles functionalized with diglycolamic acid and herceptin

Polymer mediated drug delivery system represents a novel promising platform for tumor-targeting with reduced systemic side effects and improved chemotherapeutical efficacy. In this study, we report the preparation and characterization of herceptin targeted, diglycolamic acid (DGA) functionalized polyamidoamine (PAMAM) dendrimer as a potent drug carrier for cisplatin. DGA dendrimers carrying cisplatin demonstrated enhanced anticancer activity when targeted with herceptin. In vitro cell line studies with herceptin-DGA-G4-cisplatin in HER-2 +ve and HER-2 -ve human ovarian cancer cell lines showed that these nanoparticles possessed remarkable features such as lower IC50 value, improved S-phase arrest, and enhanced apoptosis due to increased cellular uptake and accumulation than the untargeted DGA-G4-cisplatin and free cisplatin. Furthermore, in vivo results in SCID mice bearing SKOV-3 tumor xenografts, herceptin-DGA-G4-cisplatin, appeared to be more effective in inducing tumor regression as compared to free cisplatin. Collectively, these results indicate that herceptin targeted DGA functionalized PAMAM-cisplatin conjugates serve as better anti-tumor agents than individual therapeutic agents.

P21-activated kinase 1 (Pak1) signaling influences therapeutic outcome in pancreatic cancer

BACKGROUND Therapeutic resistance to gemcitabine in pancreatic ductal adenocarcinoma (PDAC) is attributed to various cellular mechanisms and signaling molecules that influence as a single factor or in combination. DESIGN In this study, utilizing in vitro p21-activated kinase 1 (Pak1) overexpression and knockdown cell line models along with in vivo athymic mouse tumor xenograft models and clinical samples, we demonstrate that Pak1 is a crucial signaling kinase in gemcitabine resistance. RESULTS Pak1 kindles resistance via modulation of epithelial-mesenchymal transition and activation of pancreatic stellate cells. Our results from gemcitabine-resistant and -sensitive cell line models showed that elevated Pak1 kinase activity is required to confer gemcitabine resistance. This was substantiated by elevated levels of phosphorylated Pak1 and ribonucleotide reductase M1 levels in the majority of human PDAC tumors when compared with normal. Delineation of the signaling pathway revealed that Pak1 confers resistance to gemcitabine by preventing DNA damage, inhibiting apoptosis and regulating survival signals via NF-κB. Furthermore, we found that Pak1 is an upstream interacting substrate of transforming growth factor β-activated kinase 1-a molecule implicated in gemcitabine resistance. Molecular mechanistic studies revealed that gemcitabine docks with the active site of Pak1; furthermore, gemcitabine treatment induces Pak1 kinase activity both in vivo and in cell-free system. Finally, results from athymic mouse tumor models illustrated that Pak1 inhibition by IPA-3 enhances the cytotoxicity of gemcitabine and brings about pancreatic tumor regression. CONCLUSION To our knowledge, this is the first study illustrating the mechanistic role of Pak1 in causing gemcitabine resistance via multiple signaling crosstalks, and hence Pak1-specific inhibitors will prove to be a better adjuvant with existing chemotherapy modality for PDAC.

Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells

ABSTRACT Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRA per se (KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential therapeutic target for modulating chemoresistance in cancer cells.