Current Molecular Combination Therapies Used for the Treatment of Breast Cancer.

Breast cancer is the second leading cause of death for women worldwide. While monotherapy (single agent) treatments have been used for many years, they are not always effective, and many patients relapse after initial treatment. Moreover, in some patients the response to therapy becomes weaker, or resistance to monotherapy develops over time. This is especially problematic for metastatic breast cancer or triple-negative breast cancer. Recently, combination therapies (in which two or more drugs are used to target two or more pathways) have emerged as promising new treatment options. Combination therapies are often more effective than monotherapies and demonstrate lower levels of toxicity during long-term treatment. In this review, we provide a comprehensive overview of current combination therapies, including molecular-targeted therapy, hormone therapy, immunotherapy, and chemotherapy. We also describe the molecular basis of breast cancer and the various treatment options for different breast cancer subtypes. While combination therapies are promising, we also discuss some of the challenges. Despite these challenges, the use of innovative combination therapy holds great promise compared with traditional monotherapies. In addition, the use of multidisciplinary technologies (such as nanotechnology and computer technology) has the potential to optimize combination therapies even further.

The Use of Nanomedicine to Target Signaling by the PAK Kinases for Disease Treatment.

P21-activated kinases (PAKs) are serine/threonine kinases involved in the regulation of cell survival, proliferation, inhibition of apoptosis, and the regulation of cell morphology. Some members of the PAK family are highly expressed in several types of cancer, and they have also been implicated in several other medical disorders. They are thus considered to be good targets for treatment of cancer and other diseases. Although there are several inhibitors of the PAKs, the utility of some of these inhibitors is reduced for several reasons, including limited metabolic stability. One way to overcome this problem is the use of nanoparticles, which have the potential to increase drug delivery. The overall goals of this review are to describe the roles for PAK kinases in cell signaling and disease, and to describe how the use of nanomedicine is a promising new method for administering PAK inhibitors for the purpose of disease treatment and research. We discuss some of the basic mechanisms behind nanomedicine technology, and we then describe how these techniques are being used to package and deliver PAK inhibitors.

Analysis of the Transcriptome: Regulation of Cancer Stemness in Breast Ductal Carcinoma In Situ by Vitamin D Compounds.

Ductal carcinoma in situ (DCIS), which accounts for one out of every five new breast cancer diagnoses, will progress to potentially lethal invasive ductal carcinoma (IDC) in about 50% of cases. Vitamin D compounds have been shown to inhibit progression to IDC in the MCF10DCIS model. This inhibition appears to involve a reduction in the cancer stem cell-like population in MCF10DCIS tumors. To identify genes that are involved in the vitamin D effects, a global transcriptomic analysis was undertaken of MCF10DCIS cells grown in mammosphere cultures, in which cancer stem-like cells grow preferentially and produce colonies by self-renewal and maturation, in the presence and absence of 1α25(OH)2D3 and a vitamin D analog, BXL0124. Using next-generation RNA-sequencing, we found that vitamin D compounds downregulated genes involved in maintenance of breast cancer stem-like cells (e.g., GDF15), epithelial-mesenchymal transition, invasion, and metastasis (e.g., LCN2 and S100A4), and chemoresistance (e.g., NGFR, PPP1R1B, and AGR2), while upregulating genes associated with a basal-like phenotype (e.g., KRT6A and KRT5) and negative regulators of breast tumorigenesis (e.g., EMP1). Gene methylation status was analyzed to determine whether the changes in expression induced by vitamin D compounds occurred via this mechanism. Ingenuity pathway analysis was performed to identify upstream regulators and downstream signaling pathway genes differentially regulated by vitamin D, including TP63 and vitamin D receptor -mediated canonical pathways in particular. This study provides a global profiling of changes in the gene signature of DCIS regulated by vitamin D compounds and possible targets for chemoprevention of DCIS progression to IDC in patients.

KPT-9274, an Inhibitor of PAK4 and NAMPT, Leads to Downregulation of mTORC2 in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is difficult to treat due to lack of druggable targets. We have found that treatment with the small molecule inhibitor KPT-9274 inhibits growth of TNBC cells and eventually leads to cell death. KPT-9274 is a dual specific inhibitor of PAK4 and Nicotinamide Phosphoribosyltransferase (NAMPT). The PAK4 protein kinase is often highly expressed in TNBC cells and has important roles in cell growth, survival, and migration. Previously we have found that inhibition of PAK4 leads to growth inhibition of TNBC cells both in vitro and in vivo. Likewise, NAMPT has been shown to be dysregulated in cancer due to its role in cell metabolism. In order to understand better how treating cells with KPT-9274 abrogates TNBC cell growth, we carried out an RNA sequencing of TNBC cells treated with KPT-9274. As a result, we identified Rictor as an important target that is inhibited in the KPT-9274 treated cells. Conversely, we found that Rictor is predicted to be activated when PAK4 is overexpressed in cells, which suggests a role for PAK4 in the regulation of Rictor. Rictor is a component of mTORC2, one of the complexes formed by the serine/threonine kinase mTOR. mTOR is important for the control of cell growth and metabolism. Our results suggest a new mechanism by which the KPT-9274 compound may block the growth of breast cancer cells, which is via inhibition of mTORC2 signaling. Consistent with this, sequencing analysis of PAK4 overexpressing cells indicates that PAK4 has a role in activation of the mTOR pathway.

P21 activated kinase signaling in cancer.

The p21 Activated Kinases (PAKs) are a family of serine threonine kinases, that consist of 6 members, PAKs 1-6, which are positioned at an intersection of multiple signaling pathways implicated in oncogenesis. The PAKs were originally identified as protein kinases that function downstream of the Ras related Rho GTPases Cdc42 and Rac. PAK1 and PAK4, which belong to Group I and Group II PAKs, respectively, are most often associated with tumorigenesis. On account of their well characterized roles in cancer, several small molecule inhibitors are being developed to inhibit the PAKs, and there is interest in investigating their efficacy as either first line or adjuvant treatments for cancer. Studies to delineate PAK regulated signaling pathways as well as the long term effects of PAK overexpression on gene expression are beginning to shed light on the mechanism by which PAK proteins may lead to cancer when they are overexpressed or activated. This review will describe the association between PAK expression in cancer, with a focus on PAK1 and PAK4, which are most often associated with the disease. The current understanding of the molecular mechanisms by which the PAKs operate in cancer will be discussed. We will also review some of the potential drug candidates, and discuss which of them are currently being tested for their efficacy in cancer treatments.

Rho GTPase effectors and NAD metabolism in cancer immune suppression.

INTRODUCTION: Sustained proliferative signaling and de-regulated cellular bioenergetics are two of the chief hallmarks of cancer. Alterations in the Ras pathway and its downstream effectors are among the major drivers for uncontrolled cell growth in many cancers. The GTPases are one of the signaling molecules that activate crucial signal transducing pathways downstream of Ras through several effector proteins. The GTPases (GTP bound) interact with several effectors and modulate a number of different biological pathways including those that regulate cytoskeleton, cellular motility, cytokinesis, proliferation, apoptosis, transcription and nuclear signaling. Similarly, the altered glycolytic pathway, the so-called 'Warburg effect', rewires tumor cell metabolism to support the biosynthetic requirements of uncontrolled proliferation. There exists strong evidence for the critical role of the glycolytic pathway's rate limiting enzymes in promoting immunosuppression. Areas covered: We review the emerging roles of GTPase effector proteins particularly the p21 activated kinase 4 (PAK4) and nicotinamide biosynthetic pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT) as signaling molecules in immune surveillance and the immune response. Expert opinion: In this expert opinion article we highlight the recent information on the role of GTPases and the metabolic enzymes on the immune microenvironment and propose some unique immune therapeutic opportunities.

Decrypting the PAK4 transcriptome profile in mammary tumor forming cells using Next Generation Sequencing.

The p-21 Activated Kinase 4 (PAK4) protein kinase is implicated in many cancers, including breast cancer. Overexpression of PAK4 is sufficient to cause mouse mammary epithelial cells (iMMECs) to become tumorigenic. To gain insight into the long-term gene expression changes that occur downstream to PAK4, we performed Next Generation Sequencing of RNA collected from PAK4 overexpressing iMMECs and wild-type iMMECs. We identified a list of genes whose expression levels were altered in response to PAK4 overexpression in iMMECs. Some of these genes, including FoxC2 and ParvB, are consistent with a role for PAK4 in cancer. In addition, PAK4 regulates many genes that are frequently associated with the inflammatory response, raising the possibility that there is a connection between PAK4, inflammation, and the tumor microenvironment. This study delineates the PAK4 transcriptome profile in transformed mammary cells and can provide translational utility in other types of cancers as well.

Pdx1-Cre-driven conditional gene depletion suggests PAK4 as dispensable for mouse pancreas development.

Constitutive depletion of p21-activated kinase 4 (PAK4) in the mouse causes embryonic lethality associated with heart and brain defects. Given that conventional gene depletion of PAK1 or PAK3 caused functional deficits in the mouse pancreas, while gene depletion of PAK5 or PAK6 did not, we asked if PAK4 might have a functional role in pancreas development. We therefore introduced conditional, Pdx1-Cre-mediated, pancreatic PAK4 gene depletion in the mouse, verified by loss of PAK4 protein expression in the pancreas. PAK4 knock-out (KO) mice were born at Mendelian ratios in both genders. Further, morphological and immunohistochemical examinations and quantifications indicated that exocrine, endocrine and ductal compartments retained the normal proportions and distributions upon PAK4 gene depletion. In addition, body weight records and a glucose tolerance test revealed no differences between WT and PAK4 KO mice. Together, this suggests that PAK4 is dispensable for mouse pancreas development. This will facilitate future use of our Pdx1-Cre-driven conditional PAK4 KO mouse model for testing in vivo potential functions of PAK4 in pancreatic disease models such as for pancreatitis and different pancreatic cancer forms.

A novel orally bioavailable compound KPT-9274 inhibits PAK4, and blocks triple negative breast cancer tumor growth.

Breast cancer is a heterogeneous disease consisting of several subtypes. Among these subtypes, triple negative breast cancer is particularly difficult to treat. This is due to a lack of understanding of the mechanisms behind the disease, and consequently a lack of druggable targets. PAK4 plays critical roles in cell survival, proliferation, and morphology. PAK4 protein levels are high in breast cancer cells and breast tumors, and the gene is often amplified in basal like breast cancers, which are frequently triple negative. PAK4 is also overexpressed in other types of cancer, making it a promising drug target. However, its inhibition is complicated by the fact that PAK4 has both kinase-dependent and -independent functions. Here we investigate a new clinical compound KPT-9274, which has been shown to inhibit PAK4 and NAMPT. We find that KPT-9274 (and its analog, KPT-8752) can reduce the steady state level of PAK4 protein in triple negative breast cancer cells. These compounds also block the growth of the breast cancer cells in vitro, and stimulate apoptosis. Most importantly, oral administration of KPT-9274 reduces tumorigenesis in mouse models of human triple negative breast cancer. Our results indicate that KPT-9274 is a novel therapeutic option for triple negative breast cancer therapy.

P21 activated kinases: structure, regulation, and functions.

The p21 activated kinases (Paks) are well known effector proteins for the Rho GTPases Cdc42 and Rac. The Paks contain 6 members, which fall into 2 families of proteins. The first family consists of Paks 1, 2, and 3, and the second consists of Paks 4, 5, and 6. While some of the Paks are ubiquitously expressed, others have more restrictive tissue specificity. All of them are found in the nervous system. Studies using cell culture, transgenic mice, and knockout mice, have revealed important roles for the Paks in cytoskeletal organization and in many aspects of cell growth and development. This review discusses the basic structures of the Paks, and their roles in cell growth, development, and in cancer.

Differential sensitivity of Pak5, Pak6, and Pak5/Pak6 double-knockout mice to the stimulant effects of amphetamine and exercise-induced alterations in body weight.

OBJECTIVES: PAK5 and PAK6 are protein kinases highly expressed in the brain. Previously, we observed that Pak6 knockout mice gained significantly more weight during development than Pak5 knockout mice as well as wild-type controls and double-knockout mice lacking both Pak5 and Pak6. In this study, we assessed the effects of exercise on food intake and weight gain of these mice as well as their sensitivity to the stimulant effects of amphetamine. METHODS: Mice of each genotype were placed in cages with free access to run wheel exercise or in cages without run wheels for a total of 74 days. Food and fluid intake as well as body weight of each mouse were measured on a weekly basis. Finally, mice were given a high dose of amphetamine and activity levels were observed immediately thereafter for 90 minutes. Brains and testes of mice were assayed for protein levels of the estrogen alpha and progesterone receptors. RESULTS: While run wheel mice consumed significantly more food, they weighed less than non-run wheel mice. In addition, although Pak6 knockout mice consumed the same amount of food as wild-type mice, they were significantly heavier regardless of run wheel condition. Pak5 knockout mice were found to be more active than other genotypes after amphetamine treatment. Finally, protein levels of the progesterone and estrogen alpha receptors were altered in brain and testes of the Pak6 knockout mice. DISCUSSION: Collectively, these data suggest that PAK6 play a role in weight gain unrelated to exercise and caloric intake and that Pak5 knockout mice are more sensitive to the stimulant effects of amphetamine.

Systems-wide analysis of K-Ras, Cdc42, and PAK4 signaling by quantitative phosphoproteomics.

Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.

PAK4-6 in cancer and neuronal development.

PAKs 4, 5 and 6 are members of the group B family of p21-activated kinases. Among this group, PAK4 has been most extensively studied. While it has essential roles in embryonic development, in adults high levels of PAK4 are frequently associated with cancer. PAK4 is overexpressed in a variety of cancers, and the Pak4 gene is amplified in some cancers. PAK4 overexpression is sufficient to cause oncogenic transformation in cells and in mouse models. The tight connection between PAK4 and cancer make it a promising diagnostic tool as well as a potential drug target. The group B PAKs also have important developmental functions. PAK4 is important for many early developmental processes, while PAK5 and PAK6 play roles in learning and memory in mice. This chapter provides an overview of the roles of the group B PAKs in cancer as well as development, and includes a discussion of PAK mediated signaling pathways and cellular functions.

The pak4 protein kinase in breast cancer.

Paks4, along with Paks5, and 6 are members of the group B family of p21-activated kinases (Paks). The Paks play multiple different roles in controlling cell morphology, cell growth, proliferation, and signaling. Pak4 has essential roles in embryonic development (Qu et al., 2003), but in adults high levels of Pak4 are frequently associated with cancer. Pak4 has been implicated in several types of cancer (Wells and Jones, 2010; Eswaran et al., 2009; Liu et al., 2008; and Liu et al., 2010) and it is strongly linked to breast cancer (Liu et al., 2008; Liu et al. 2010; Yu et al., 2009; Rafn et al., 2012; and So et al., 2012). Breast tumors and breast cancer cell lines frequently have high levels of Pak4 (Liu et al., 2008), and overexpression of Pak4 in mammary epithelial cells leads to tumorigenesis in mice (Liu et al., 2010). This paper summarizes the current work on the role of Pak4 in breast cancer.

Differential Expression of Key Signaling Proteins in MCF10 Cell Lines, a Human Breast Cancer Progression Model.

Breast cancer is a heterogeneous disease that develops through a multistep process whose molecular basis remains poorly understood. The molecular mechanisms of breast cancer progression have been extensively studied using the MCF10 model. We summarized recent results on differential expression of proteins in the MCF10 cell series - MCF10A, MCF10AT1, MCF10DCIS.com and MCF10CA1a - and compared the ability of the latter 3 lines to form tumors in immunodeficient mice. In addition, we also investigated expression of several key signaling proteins in the MCF10 cell series corresponding to different stages of breast cancer progression. MCF10DCIS.com and MCF10CA1a cells were highly tumorigenic; MCF10CA1a cells showed more aggressive tumor growth than MCF10DCIS.com cells. HRAS-driven cancer initiation stage was accompanied by the increased expression of c-Myc, cyclin D1 and IGF-IR. Tumorigenic cell lines expressed higher levels of pErk, pAkt, Stat3 and Pak4 compared to nontumorigenic cells. The expression of CD44v, CD44v3, CD44v6, ERBB2, Cox2 and Smad4 correlated with the increased tumorigenicity of the MCF10 cell lines. The differences in expression of signaling proteins involved in breast cancer progression may provide new insight into the mechanisms of tumorigenesis and useful information for development of targeted therapeutics.

PAK4 is required for regulation of the cell-cycle regulatory protein p21, and for control of cell-cycle progression.

The serine/threonine kinase PAK4 regulates cytoskeletal architecture, and controls cell proliferation and survival. In most adult tissues PAK4 is expressed at low levels, but overexpression of PAK4 is associated with uncontrolled proliferation, inappropriate cell survival, and oncogenic transformation. Here we have studied for the first time, the role for PAK4 in the cell cycle. We found that PAK4 levels peak dramatically but transiently in the early part of G1 phase. Deletion of Pak4 was also associated with an increase in p21 levels, and PAK4 was required for normal p21 degradation. In serum-starved cells, the absence of PAK4 led to a reduction in the amount of cells in G1, and an increase in the amount of cells in G2/M phase. We propose that the transient increase in PAK4 levels at early G1 reduces p21 levels, thereby abrogating the activity of CDK4/CDK6 kinases, and allowing cells to proceed with the cell cycle in a precisely coordinated way.

p120-catenin is a binding partner and substrate for Group B Pak kinases.

Pak5 is a member of the Group B p21-activated kinases, which are effectors of the Rho family GTPases Cdc42 and Rac. Pak5 has been shown to promote cytoskeletal reorganization, inducing filopodia formation and neurite outgrowth in neuroblastoma cells. In this study, we used affinity chromatography followed by SDS-PAGE and mass spectrometry to identify potential downstream effectors of Pak5. Using this approach, we isolated p120-catenin (p120), a known regulator of cytoskeletal reorganization and Rho GTPases. Using co-immunoprecipitation assays we found that p120 preferentially interacts with Pak5 among the Group B Paks. Results from immunofluorescence studies revealed that Pak5 and p120 co-localize in cells. Both Pak5 and constitutively active Pak4, the founding member of the Group B Paks, directly phosphorylate p120 in vitro. The phosphorylation was shown by Western blot and immunofluorescence to take place specifically on serine 288. This study is the first report of an upstream serine/threonine kinase that phosphorylates p120.

The pak4 protein kinase plays a key role in cell survival and tumorigenesis in athymic mice.

Pak4 is a member of the B group of p21-activated (Pak) kinases, originally identified as an effector protein for Cdc42. Although Pak4 is expressed at low levels in most adult tissues, it is highly overexpressed in tumor cell lines. Here, we show that Pak4 is also overexpressed in primary tumors, including colon, esophageal, and mammary tumors. Overexpression of Pak4 also leads to tumor formation in athymic mice, whereas deletion of Pak4 inhibits tumorigenesis. Although a constitutively active Pak4 mutant was previously shown to promote oncogenic transformation in cultured cells, our results are the first to show that Pak4 also promotes tumorigenesis in experimental animals. Furthermore, these results show for the first time that not only constitutively active Pak4, but also wild-type Pak4, is transforming, when experimental animals are used. These results are highly significant because wild-type Pak4, rather than activated Pak4, is overexpressed in tumor cells. Our results suggest that overexpression or activation of Pak4 is a key step in oncogenic transformation, due to its ability to promote cell survival and subsequent uncontrolled proliferation. The finding that Pak4 is up-regulated in so many types of cancers indicates that Pak4 may play a vital role in a wide range of different types of cancer. This makes it an attractive candidate for drug therapy for different types of cancer.

PAK4 and alphaPIX determine podosome size and number in macrophages through localized actin regulation.

Podosomes are actin-rich adhesion structures typical for monocytic cells and are implicated in migration and invasion. Major modes of podosome regulation include RhoGTPase signaling and actin regulatory pathways. However, it is not clearly understood how these signals induce highly localized changes in podosome formation and dynamics. Here, we show that the RhoGTPase effector PAK4, a member of the p21 associated kinase family, and its regulator alphaPIX (PAK-interacting exchange factor), are central to podosome formation in primary human macrophages. Immunofluorescence, biochemical and microarray data indicate that PAK4 acts as physiological regulator of podosomes in this system. Accordingly, transfection of a specific shRNA, as well as expression of PAK4 truncation mutants, resulted in reduced numbers of podosomes per cell. Moreover, expression of kinase active or inactive PAK4 mutants enhanced or reduced the size of individual podosomes, respectively, indicating a modulatory influence of PAK4 kinase activity on podosome size. Similar to the results gained with PAK4, cellular/overexpressed PIX was shown to be closely associated with podosomes. Moreover, both overexpression of alphaPIX wt and a mutant lacking the SH3 domain led to coalescence of podosomes. In sum, we propose that PAK4 and alphaPIX can induce highly localized changes in actin dynamics and thereby regulate size and number of podosomes in primary human macrophages.

Pak4 induces premature senescence via a pathway requiring p16INK4/p19ARF and mitogen-activated protein kinase signaling.

Exposure of primary cells to mitogenic stimuli or oncogenes often causes them to undergo premature senescence. This is most likely a protective function that prevents uncontrolled proliferation. Pak4 is a target for the Rho GTPase Cdc42. Pak4 is overexpressed in human tumor cell lines, and it is the only member of the Pak family that is highly transforming in immortalized fibroblasts. Here we show that in primary fibroblasts, activated Pak4 inhibits cell proliferation and promotes premature senescence. Furthermore, Pak4 expression levels are upregulated in response to stimuli that promote senescence. Pak4-induced arrest appears to be mediated by a pathway that requires the ERK mitogen-activated protein kinase, as well as the cell cycle inhibitors p16(INK4) and p19(ARF). These new results describing a role for Pak4 in senescence are important for understanding why this protein is associated with cancer and how it promotes transformation in immortalized cells.