Melody Stallings-Mann

A novel small-molecule inhibitor of protein kinase Cι blocks transformed growth of non-small-cell lung cancer cells

We recently showed that atypical protein kinase Cι (PKCι) is required for transformed growth of human non–small-cell lung cancer (NSCLC) cells by activating Rac1. Genetic disruption of PKCι signaling blocks Rac1 activity and transformed growth, indicating that PKCι is a viable target for development of novel therapeutics for NSCLC. Here, we designed and implemented a novel fluorescence resonance energy transfer–based assay to identify inhibitors of oncogenic PKCι signaling. This assay was used to identify compounds that disrupt the interaction between PKCι and its downstream effector Par6, which links PKCι to Rac1. We identified aurothioglucose (ATG), a gold compound used clinically to treat rheumatoid arthritis, and the related compound, aurothiomalate (ATM), as potent inhibitors of PKCι-Par6 interactions in vitro (IC50 ∼1 μmol/L). ATG blocks PKCι-dependent signaling to Rac1 and inhibits transformed growth of NSCLC cells. ATG-mediated inhibition of transformation is relieved by expression of constitutively active Rac1, consistent with a mechanism at the level of the interaction between PKCι and Par6. ATG inhibits A549 cell tumor growth in nude mice, showing efficacy against NSCLC in a relevant preclinical model. Our data show the utility of targeting protein-protein interactions involving PKCι for antitumor drug development and provide proof of concept that chemical disruption of PKCι signaling can be an effective treatment for NSCLC. ATG and ATM will be useful reagents for studying PKCι function in transformation and represent promising new agents for the clinical treatment of NSCLC. (Cancer Res 2006; 66(3): 1767-74)

Aurothiomalate Inhibits Transformed Growth by Targeting the PB1 Domain of Protein Kinase Cι

We recently identified the gold compound aurothiomalate (ATM) as a potent inhibitor of the Phox and Bem1p (PB1)-PB1 domain interaction between protein kinase C (PKC) ι and the adaptor molecule Par6. ATM also blocks oncogenic PKCι signaling and the transformed growth of human lung cancer cells. Here we demonstrate that ATM is a highly selective inhibitor of PB1-PB1 domain interactions between PKCι and the two adaptors Par6 and p62. ATM has no appreciable inhibitory effect on other PB1-PB1 domain interactions, including p62-p62, p62-NBR1, and MEKK3-MEK5 interactions. ATM can form thio-gold adducts with cysteine residues on target proteins. Interestingly, PKCι (and PKCζ) contains a unique cysteine residue, Cys-69, within its PB1 domain that is not present in other PB1 domain containing proteins. Cys-69 resides within the OPR, PC, and AID motif of PKCι at the binding interface between PKCι and Par6 where it interacts with Arg-28 on Par6. Molecular modeling predicts formation of a cysteinyl-aurothiomalate adduct at Cys-69 that protrudes into the binding cleft normally occupied by Par6, providing a plausible structural explanation for ATM inhibition. Mutation of Cys-69 of PKCι to isoleucine or valine, residues frequently found at this position in other PB1 domains, has little or no effect on the affinity of PKCι for Par6 but confers resistance to ATM-mediated inhibition of Par6 binding. Expression of the PKCι C69I mutant in human non-small cell lung cancer cells confers resistance to the inhibitory effects of ATM on transformed growth. We conclude that ATM inhibits cellular transformation by selectively targeting Cys-69 within the PB1 domain of PKCι.

Single proteins might have dual but related functions in intracellular and extracellular microenvironments.

The maintenance of organ homeostasis and the control of an appropriate response to environmental alterations require the intimate coordination of cellular functions and tissue organization. An important component of this coordination could be provided by proteins that can have distinct but linked functions on both sides of the plasma membrane. We present a model that proposes that unconventional secretion provides a mechanism through which single proteins can integrate complex tissue functions.

Enzyme replacement therapy results in substantial improvements in early clinical phenotype in a mouse model of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a devastating, degenerative neurological disorder caused by mutations in the galactosylceramidase (GALC) gene that severely affect enzyme activity. Currently, treatment options for this disorder are very limited. Enzyme replacement therapy (ERT) has been shown to be effective in lysosomal storage disorders with predominantly peripheral manifestations such as type I Gauchers and Fabrys disease. Little however is known about the possible benefit of ERT in GLD, which has a substantial central nervous system component. In this study, we examined the effect of peripheral GALC injections in the twitcher mouse model of the disease. Although we were unable to block the precipitous decline that normally occurs just before death, we did observe significant early improvements in motor performance, a substantial attenuation in the initial failure to thrive, and an increase in life span. Immunohistochemical and activity analyses demonstrated GALC uptake in multiple tissues, including the brain. This was associated with a decrease in the abnormal accumulation of the GALC substrate psychosine, which is thought to play a pivotal role in disease pathology. These results indicate that peripheral ERT is likely to be beneficial in GLD.

Matrix Metalloproteinase Induction of Rac1b, a Key Effector of Lung Cancer Progression

Rac1b mediates the matrix metalloproteinase–induced epithelial-mesenchymal transition in lung adenocarcinoma and is a potential therapeutic target in early-stage lung cancer. Neutralizing the Neighbors Life-long addictions to cigarettes—and the accompanying enhanced risk of lung cancer—often begin with a succumbing to peer pressure. Now, Stallings-Mann et al. characterize lung cancer–related peer pressure at the molecule level by showing that a protease in the extracellular matrix compels expression, in neighboring lung cells, of a signaling protein (Rac1B) that acts as a mediator of lung cancer progression. Exposure to cigarette smoke is the key cause of non–small cell lung cancers (NSCLCs), which constitute >80% of all cases of lung cancer. NSCLC is most effectively treated when detected early, but to stop this cancer in its tracks, scientists must learn more about the molecular mechanisms of tumor progression and then use this information to discover new targeted therapies. Rac1b is a tumor-associated, cell-transforming protein that arises as an alternatively spliced isoform of Rac1, a Rho family GTPase that regulates cell proliferation. It has been hypothesized that Rac1b drives oncogenesis by promoting epithelial-mesenchymal transition (EMT), during which epithelial cells detach from each other and from the underlying basement membrane and acquire invasive properties. Matrix metalloproteinases (MMPs) live in and cleave components of the extracellular matrix that borders epithelial cells and have been shown to induce EMT. Stallings-Mann et al. found that expression of mouse Rac1b in lung epithelial cells of transgenic mice spurred both EMT and spontaneous tumor formation. Furthermore, in the transgenic mice, MMP3-induced expression of Rac1b stimulated EMT and progression of the premalignant state in lung epithelial cells to malignant lung adenocarcinoma by bypassing oncogene-induced senescence. In patients, Rac1b mRNA expression was elevated in lung adenocarcinoma relative to adjacent normal tissue, in stage-2 relative to stage-1 lung tumors, and in normal tissue from smokers compared to nonsmokers. How MMP3, Rac1b, and EMT collaborate to drive tumor progression remains unclear. But the new work suggests that drugs that block synthesis or function of Rac1b—which has no known function in normal cells—may prevent progression to late-stage, invasive forms of lung cancer. Lung cancer is more deadly than colon, breast, and prostate cancers combined, and treatment improvements have failed to improve prognosis significantly. Here, we identify a critical mediator of lung cancer progression, Rac1b, a tumor-associated protein with cell-transforming properties that are linked to the matrix metalloproteinase (MMP)–induced epithelial-mesenchymal transition (EMT) in lung epithelial cells. We show that expression of mouse Rac1b in lung epithelial cells of transgenic mice stimulated EMT and spontaneous tumor development and that activation of EMT by MMP-induced expression of Rac1b gave rise to lung adenocarcinoma in the transgenic mice through bypassing oncogene-induced senescence. Rac1b is expressed abundantly in stages 1 and 2 of human lung adenocarcinomas and, hence, is an attractive molecular target for the development of new therapies that prevent progression to later-stage lung cancers.

Spontaneous activation and signaling by overexpressed epidermal growth factor receptors in glioblastoma cells.

Overexpressed epidermal growth receptor factor receptors (EGFRs) are thought to contribute to the malignant phenotype of human glioblastomas (GBMs), but the mechanism is not well understood. We found that SKMG‐3 cells, a rare GBM cell line that maintains EGFR gene amplification in vitro, produced high levels of EGFR protein. The cells also expressed the related receptors HER2/neu and HER4, but not HER3. Immunoblots and tryptic phosphopeptide maps showed that the SKMG‐3 EGFRs were intact and functional and that a subset of these receptors were spontaneously autophosphorylated. EGF treatment stimulated phosphorylation of the EGFRs as well as the downstream effectors Erk, AKT1, stat3 and c‐Cbl. Under minimal growth conditions, the unstimulated SKMG‐3 cells contained constitutively phosphorylated Erk and AKTI but no detectable stat3 DNA‐binding complexes. The EGFR kinase inhibitor PD158780 reduced the constitutive phosphorylation of the receptor and Erk but not that of AKT1. In contrast, inhibition of phosphatidylinositol‐3‐kinase (PI3K) blocked the constitutive phosphorylation of Erk and AKT‐1 but not the EGFR. We conclude that the SKMG‐3 cells represent the subset of GBMs with amplified EGFR genes that overexpress intact receptors. The results also suggest that in some GBMs, signals from overexpressed EGFRs contribute to the constitutive phosphorylation of Erk, but these signals may not required for the constitutive activation of PI3K or AKT1.

Matrix metalloproteinase-induced malignancy in mammary epithelial cells.

In development, epithelial-mesenchymal transition (EMT) allows interconnected sheets of epithelial cells to reorganize and to pass into and through the surrounding extracellular matrix (ECM). In cancer, EMT-associated processes facilitate invasive growth and development of metastases. Matrix metalloproteinases (MMPs) are enzymes capable of degrading the ECM and altering cell-cell and cell-ECM interactions. MMPs are upregulated in nearly all tumor types, have been shown to induce EMT in cultured cells, and are involved in the development of tumor formation, invasion, and metastasis. We have identified the induction of Rac1b, an alternatively spliced variant of Rac1, as a key event in MMP-induced EMT. Induction of the Rac1b isoform leads to increased cellular reactive oxygen species (ROS), which causes in turn upregulation of Snail, a transcription factor previously implicated in physiological and pathological EMT. MMP/Rac1b-induced ROS also causes DNA damage and induces genomic instability. These findings reveal a new pathway in which a key element of the tumor microenvironment directly stimulates the phenotypic and genotypic alterations involved in malignant transformation, and provides many opportunities for investigation of therapeutic interventions.

Involvement of hnRNP A1 in the matrix metalloprotease‐3‐dependent regulation of Rac1 pre‐mRNA splicing

Rac1b is an alternatively spliced isoform of the small GTPase Rac1 that includes the 57‐nucleotide exon 3b. Rac1b was originally identified through its over‐expression in breast and colorectal cancer cells, and has subsequently been implicated as a key player in a number of different oncogenic signaling pathways, including tumorigenic transformation of mammary epithelial cells exposed to matrix metalloproteinase‐3 (MMP‐3). Although many of the cellular consequences of Rac1b activity have been recently described, the molecular mechanism by which MMP‐3 treatment leads to Rac1b induction has not been defined. Here we use proteomic methods to identify heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a factor involved in Rac1 splicing regulation. We find that hnRNP A1 binds to Rac1 exon 3b in mouse mammary epithelial cells, repressing its inclusion into mature mRNA. We also find that exposure of cells to MMP‐3 leads to release of hnRNP A1 from exon 3b and the consequent generation of Rac1b. Finally, we analyze normal breast tissue and breast cancer biopsies, and identify an inverse correlation between expression of hnRNP A1 and Rac1b, suggesting the existence of this regulatory axis in vivo. These results provide new insights on how extracellular signals regulate alternative splicing, contributing to cellular transformation and development of breast cancer. J. Cell. Biochem. 113: 2319–2329, 2012.

Immune cell quantitation in normal breast tissue lobules with and without lobulitis

While the immune microenvironment has been investigated in breast cancers, little is known about its role in non-malignant breast tissues. Here we quantify and localize cellular immune components in normal breast tissue lobules, with and without visible immune infiltrates (lobulitis). Up to ten representative lobules each in eleven normal breast tissue samples were assessed for B cells (CD20), cytotoxic T cells (CD8), helper T cells (CD4), dendritic cells (CD11c), leukocytes (CD45), and monocytes/macrophages (CD68). Using digital image analysis, immune cell densities were measured and compared between lobules with/without lobulitis. 109 lobules in 11 normal breast tissue samples were evaluated; 31 with lobulitis and 78 without. Immune cells showed consistent patterns in all normal samples, predominantly localized to lobules rather than stroma. Regardless of lobulitis status, most lobules demonstrated CD8+, CD11c+, CD45+, and CD68+ cells, with lower densities of CD4+ and CD20+ cells. Both CD11c+ and CD8+ cells were consistently and intimately associated with the basal aspect of lobule epithelium. Significantly higher densities of CD4+, CD8+, CD20+, and CD45+ cells were observed in lobules with lobulitis. In contrast, densities of monocytes/macrophages and dendritic cells did not vary with lobulitis. In normal breast tissue, myeloid and lymphoid cells are present and localized to lobules, with cytotoxic T and dendritic cells directly integrated with epithelium. Lobules with lobulitis have significantly more adaptive immune (B and T) cells, but no increase in dendritic cells or monocytes/macrophages. These findings indicate an active and dynamic mucosal immune system in normal breast tissue.

Tissue Stiffness and Hypoxia Modulate the Integrin-Linked Kinase ILK to Control Breast Cancer Stem-like Cells

Breast tumors are stiffer and more hypoxic than nonmalignant breast tissue. Here we report that stiff and hypoxic microenvironments promote the development of breast cancer stem-like cells (CSC) through modulation of the integrin-linked kinase ILK. Depleting ILK blocked stiffness and hypoxia-dependent acquisition of CSC marker expression and behavior, whereas ectopic expression of ILK stimulated CSC development under softer or normoxic conditions. Stiff microenvironments also promoted tumor formation and metastasis in ovo, where depleting ILK significantly abrogated the tumorigenic and metastatic potential of invasive breast cancer cells. We further found that the ILK-mediated phenotypes induced by stiff and hypoxic microenvironments are regulated by PI3K/Akt. Analysis of human breast cancer specimens revealed an association between substratum stiffness, ILK, and CSC markers, insofar as ILK and CD44 were expressed in cancer cells located in tumor regions predicted to be stiff. Our results define ILK as a key mechanotransducer in modulating breast CSC development in response to tissue mechanics and oxygen tension. Cancer Res; 76(18); 5277-87. ©2016 AACR.