Src, PKCα, and PKCδ are required for αvβ3 integrin-mediated metastatic melanoma invasion

<p>Abstract</p> <p>Background</p> <p>Integrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor αvβ3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis.</p> <p>Results</p> <p>Two melanoma cell lines C8161.9 and M14 both express high levels of αvβ3 integrin and adhere to vitronectin. However, only the highly metastatic C8161.9 cells are capable of invading vitronectin-enriched Matrigel in an αvβ3-depenent manner. Elevated levels of PKCα and PKCδ, and activated Src were detected specifically in the highly metastatic melanoma cells, but not in the low metastatic M14 cells. Inhibition of Src or PKC activity suppressed αvβ3-dependent invasion. Furthermore, over expression of Src or PKCα and PKCδ was sufficient to confer αvβ3-dependent invasiveness to M14 cells. Stress fiber formation and focal adhesion formation were almost completely absent in C8161.9 cells compared to M14 cells. Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity. Src had no effect on Rac activity. Loss of PKCα expression, but not PKCδ, by siRNA inhibited Rac and PAK activity as well as invasiveness. Loss of PKCα restored focal adhesion formation and partially restored stress fiber formation, while loss of PKCδ primarily restored stress fibers.</p> <p>Conclusion</p> <p>The misregulated expression of PKCα and PKCδ and elevated Src activity in metastatic melanoma cells is required for efficient αvβ3-mediated invasion. PKCα and Src enhance αvβ3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA. PKCα influences focal adhesion formation, while PKCδ controls stress fibers.</p

Cancer Cell-Extrinsic Roles for the Androgen Receptor in Prostate Cancer

Given the central role of the androgen receptor (AR) in prostate cancer cell biology, AR-targeted therapies have been the backbone of prostate cancer treatment for over 50 years. New data indicate that AR is expressed in additional cell types within the tumor microenvironment. Moreover, targeting AR for the treatment of prostate cancer has established side effects such as bone complications and an increased risk of developing cardiometabolic disease, indicating broader roles for AR. With the advent of novel technologies, such as single-cell approaches and advances in preclinical modeling, AR has been identified to have clinically significant functions in other cell types. In this mini-review, we describe new cancer cell-extrinsic roles for AR within the tumor microenvironment as well as systemic effects that collectively impact prostate cancer progression and patient outcomes

Activation of Syk protein tyrosine kinase through interaction with integrin β cytoplasmic domains

AbstractSyk protein tyrosine kinase is essential for immune system development and function [1] and for the maintenance of vascular integrity [2, 3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs) [1]. Syk can also be activated by integrin adhesion receptors [4, 5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin β3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the β3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the β3 cytoplasmic tail [β3(759X)] decreased Syk binding and disrupted its physical association with integrin αIIbβ3. Furthermore, cells expressing αIIbβ3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the αIIbβ3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin β3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase

A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector

Background: Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. Methods: First, we modified the Tet-pLKO-Puro vector to make it easy("EZ") for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods. Results: Notably, we emphasize the importance of loop sequence design and demonstrate that the addition of a single mismatch in the loop stem can greatly improve shRNA efficiency. Lastly, we display the robustness of the system with a doxycycline titration and recovery time course and provide a cost/benefit analysis comparing our system with purchasing pre-designed shRNA vectors. Conclusions: Our aim was twofold: first, to take a very useful shRNA vector and make it more amenable for molecular cloning and, secondly, to provide a streamlined protocol and rationale for cost-effective design, cloning, and screening of shRNAs. With this knowledge, anyone can take advantage of this powerful tool to inducibly knockdown any gene of their choosing.National Cancer Institute of the National Institutes of Health [R01CA154835, P30CA023074]; Department of Defense [W81XWH-14-1-0479]; Worldwide Cancer Fund [11-0082]; Van Andel Research Institute; University of ArizonaOpen Access JournalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Disruption of prostate epithelial differentiation pathways and prostate cancer development

A major problem in the prostate cancer field is the inability to distinguish aggressive from indolent disease. This gap in knowledge stems from the fact that the mechanisms which trigger tumorigenesis in the prostate are poorly understood. While specific known genetic alterations are very common in prostate tumors, including Myc upregulation, fusion of Ets genes to androgen-regulated promoters, and loss of Pten, the specific roles of these aberrations in prostate tumor initiation and progression are poorly understood. Likewise, the cell of origin for prostate cancer remains controversial and may be linked to the aggressive potential of the tumor. One important clue about the oncogenic cell of origin is that prostate tumors coexpress basal and luminal protein markers normally restricted to their respective cell types in the prostate epithelia. We hypothesize this coexpression is indicative of the fact that prostate tumors arise as a defect in normal epithelial differentiation. Moreover, we hypothesize that rare bipotent cells in the prostate, which can give rise to basal or luminal cells, are particularly sensitive to oncogenic conversion and are a cell of origin for prostate cancer. In support of this hypothesis, many of the pathways involved in prostate differentiation are linked to genes commonly altered in prostate cancer. In this article, we review what is known about important differentiation pathways in the prostate (Myc, p38MAPK, Notch, PI3K/Pten) and how their misregulation in a transiently differentiating bipotent epithelial cell could lead to oncogenesis. Better understanding of normal prostate differentiation will offer new insights into tumor initiation and may help to explain the functional significance of common genetic alterations observed in prostate cancer. Additionally, this understanding could lead to new methods for classifying prostate tumors based on their differentiation status and may aid in identifying more aggressive tumors

Human prostate luminal cell differentiation requires NOTCH3 induction by p38-MAPK and MYC

Many pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an in vitro differentiation model, we tested the hypothesis that regulation of NOTCH3 by the p38 MAPK family (hereafter p38-MAPK), via MYC, is required for luminal differentiation. Inhibition (SB202190 and BIRB796) or knockdown of p38a (also known as MAPK14) and/or p38d (also known as MAPK13) prevented proper differentiation. Additionally, treatment with a gamma-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation through MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA and protein levels, which was diminished upon MYC inhibition (10058-F4 and JQ1) or knockdown. Furthermore, we validated two NOTCH3 enhancer elements through a combination of enhancer (e) RNA detection (BruUV-seq) and luciferase reporter assays. Finally, we found that the NOTCH3 mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC and NOTCH signaling, demonstrate two mechanisms of NOTCH3 regulation and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.U.S. Department of Defense Prostate Cancer Research Program [W81XWH-14-1-0479]; Association for International Cancer Research (Worldwide Cancer Research) [11-0082]; Van Andel Research Institute; University of Arizona12 month embargo; Published online June 1, 2017.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Global deletion of tetraspanin CD82 attenuates bone growth and enhances bone marrow adipogenesis

CD82 is a widely expressed member of the tetraspanin family of transmembrane proteins known to control cell signaling, adhesion, and migration. Tetraspanin CD82 is induced over 9-fold during osteoclast differentiation in vitro; however, its role in bone homeostasis is unknown. A globally deleted CD82 mouse model was used to assess the bone phenotype. Based on microCT and 4-point bending tests, CD82-deficient bones are smaller in diameter and weaker, but display no changes in bone density. Histomorphometry shows a decrease in size, erosion perimeter, and number of osteoclasts in situ, with a corresponding increase in trabecular surface area, specifically in male mice. Male-specific alterations are observed in trabecular structure by microCT and in vitro differentiated osteoclasts are morphologically abnormal. Histomorphometry did not reveal a significant reduction in osteoblast number; however, dynamic labeling reveals a significant decrease in bone growth. Consistent with defects in OB function, OB differentiation and mineralization are defective in vitro, whereas adipogenesis is enhanced. There is a corresponding increase in bone marrow adipocytes in situ. Thus, combined defects in both osteoclasts and osteoblasts can account for the observed bone phenotypes, and suggests a role for CD82 in both bone mesenchyme and myeloid cells.Van Andel Research Institute; Van Andel Institute Graduate School12 month embargo; published online: 18 May 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Src, PKCalpha, and PKCdelta are required for alphavbeta integrin-mediated metastatic melanoma invasion

Background: Integrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor alpha v beta 3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis. Results: Two melanoma cell lines C8161.9 and M14 both express high levels of alpha v beta 3 integrin and adhere to vitronectin. However, only the highly metastatic C8161.9 cells are capable of invading vitronectin-enriched Matrigel in an alpha v beta 3-depenent manner. Elevated levels of PKC alpha and PKC delta, and activated Src were detected specifically in the highly metastatic melanoma cells, but not in the low metastatic M14 cells. Inhibition of Src or PKC activity suppressed alpha v beta 3-dependent invasion. Furthermore, over expression of Src or PKCa and PKCd was sufficient to confer alpha v beta 3-dependent invasiveness to M14 cells. Stress fiber formation and focal adhesion formation were almost completely absent in C8161.9 cells compared to M14 cells. Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity. Src had no effect on Rac activity. Loss of PKC alpha expression, but not PKC delta, by siRNA inhibited Rac and PAK activity as well as invasiveness. Loss of PKC alpha restored focal adhesion formation and partially restored stress fiber formation, while loss of PKC delta primarily restored stress fibers. Conclusion: The misregulated expression of PKC alpha and PKC beta and elevated Src activity in metastatic melanoma cells is required for efficient alpha v beta 3-mediated invasion. PKC alpha and Src enhance alpha v beta 3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA. PKC alpha influences focal adhesion formation, while PKCd controls stress fibers