Teresa Knifley

LPA, HGF, and EGF utilize distinct combinations of signaling pathways to promote migration and invasion of MDA-MB-231 breast carcinoma cells

BackgroundVarious pathways impinge on the actin-myosin pathway to facilitate cell migration and invasion including members of the Rho family of small GTPases and MAPK. However, the signaling components that are considered important for these processes vary substantially within the literature with certain pathways being favored. These distinctions in signaling pathways utilized are often attributed to differences in cell type or physiological conditions; however, these attributes have not been systematically assessed.MethodsTo address this question, we analyzed the migration and invasion of MDA-MB-231 breast carcinoma cell line in response to various stimuli including lysophosphatidic acid (LPA), hepatocyte growth factor (HGF) and epidermal growth factor (EGF) and determined the involvement of select signaling pathways that impact myosin light chain phosphorylation.ResultsLPA, a potent stimulator of the Rho-ROCK pathway, surprisingly did not require the Rho-ROCK pathway to stimulate migration but instead utilized Rac and MAPK. In contrast, LPA-stimulated invasion required Rho, Rac, and MAPK. Of these three major pathways, EGF-stimulated MDA-MB-231 migration and invasion required Rho; however, Rac was essential only for invasion and MAPK was dispensable for migration. HGF signaling, interestingly, utilized the same pathways for migration and invasion, requiring Rho but not Rac signaling. Notably, the dependency of HGF-stimulated migration and invasion as well as EGF-stimulated invasion on MAPK was subject to the inhibitors used. As expected, myosin light chain kinase (MLCK), a convergence point for MAPK and Rho family GTPase signaling, was required for all six conditions.ConclusionsThese observations suggest that, while multiple signaling pathways contribute to cancer cell motility, not all pathways operate under all conditions. Thus, our study highlights the plasticity of cancer cells to adapt to multiple migratory cues.

S100A4 drives non-small cell lung cancer invasion, associates with poor prognosis, and is effectively targeted by the FDA-approved anti-helminthic agent niclosamide

S100A4 (metastasin-1), a metastasis-associated protein and marker of the epithelial to mesenchymal transition, contributes to several hallmarks of cancer and has been implicated in the progression of several types of cancer. However, the impacts of S100A4 signaling in lung cancer progression and its potential use as a target for therapy in lung cancer have not been properly explored. Using established lung cancer cell lines, we demonstrate that S100A4 knockdown reduces cell proliferation, invasion and three-dimensional invasive growth, while overexpression of S100A4 increases invasive potential. In patient-derived tissues, S100A4 is preferentially elevated in lung adenocarcinoma. This elevation is associated with lymphovascular invasion and decreased overall survival. In addition, depletion of S100A4 by shRNA inhibits NF-κB activity and decreases TNFα-induced MMP9 expression. Furthermore, inhibition of the NF-κB/MMP9 axis decreases lung carcinoma invasive potential. Niclosamide, a reported inhibitor of S100A4, blocks expression and function of S100A4 with a reduction in proliferation, invasion and NF-κB-mediated MMP9 expression. Collectively, this study highlights the importance of the S100A4/NF-κB/MMP9 axis in lung cancer invasion and provides a rationale for targeting S100A4 to combat lung cancer.

Integrin α6β4 Promotes Autocrine Epidermal Growth Factor Receptor (EGFR) Signaling to Stimulate Migration and Invasion toward Hepatocyte Growth Factor (HGF).

Background: Integrin α6β4 is overexpressed in pancreatic cancer and enhances invasion. Results: Integrin α6β4 coordinately up-regulates AREG, EREG, and MMP1 through DNA demethylation and NFAT5 that in turn enhances HGF-mediated invasion. Conclusion: Integrin α6β4 stimulates HGF-dependent invasion through autocrine EGFR signaling. Significance: HGF-stimulated invasion is dependent on autocrine EGFR signaling, thus implicating why EGFR inhibitors are effective in a complex tumor microenvironment. Integrin α6β4 is up-regulated in pancreatic adenocarcinomas where it contributes to carcinoma cell invasion by altering the transcriptome. In this study, we found that integrin α6β4 up-regulates several genes in the epidermal growth factor receptor (EGFR) pathway, including amphiregulin (AREG), epiregulin (EREG), and ectodomain cleavage protease MMP1, which is mediated by promoter demethylation and NFAT5. The correlation of these genes with integrin α6β4 was confirmed in The Cancer Genome Atlas Pancreatic Cancer Database. Based on previous observations that integrin α6β4 cooperates with c-Met in pancreatic cancers, we examined the impact of EGFR signaling on hepatocyte growth factor (HGF)-stimulated migration and invasion. We found that AREG and EREG were required for autocrine EGFR signaling, as knocking down either ligand inhibited HGF-mediated migration and invasion. We further determined that HGF induced secretion of AREG, which is dependent on integrin-growth factor signaling pathways, including MAPK, PI3K, and PKC. Moreover, matrix metalloproteinase activity and integrin α6β4 signaling were required for AREG secretion. Blocking EGFR signaling with EGFR-specific antibodies or an EGFR tyrosine kinase inhibitor hindered HGF-stimulated pancreatic carcinoma cell chemotaxis and invasive growth in three-dimensional culture. Finally, we found that EGFR was phosphorylated in response to HGF stimulation that is dependent on EGFR kinase activity; however, c-Met phosphorylation in response to HGF was unaffected by EGFR signaling. Taken together, these data illustrate that integrin α6β4 stimulates invasion by promoting autocrine EGFR signaling through transcriptional up-regulation of key EGFR family members and by facilitating HGF-stimulated EGFR ligand secretion. These signaling events, in turn, promote pancreatic carcinoma migration and invasion.

Use of Synthetic Isoprenoids to Target Protein Prenylation and Rho GTPases in Breast Cancer Invasion

Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper subcellular localization to cellular membranes, which is regulated by a series of post-translational modifications that result in either farnesylation or geranylgeranylation of the C-terminal CAAX motif. This concept provided the rationale for targeting farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) for cancer treatment. However, the resulting prenyl transferase inhibitors have not performed well in the clinic due to issues with alternative prenylation and toxicity. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are farnesol or geranyl-geraniol analogs that act as alternate substrates for FTase or GGTase. Here, we test the ability of our lead PFIs, anilinogeraniol (AGOH) and anilinofarnesol (AFOH), to block the invasion of breast cancer cells. We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D) invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D) culture model at as little as 20 µM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase. To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA and RhoC activation and invasive growth. Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells. Collectively, this study demonstrates that AGOH and AFOH dramatically inhibit breast cancer invasion, at least in part by blocking Rho function, thus, suggesting that targeting prenylation by using PFIs may offer a promising mechanism for treatment of invasive breast cancer.

Abstract A44: S100A4 drives the invasive potential of non-small cell lung cancer cells and is targeted by FDA approved drugs

The metastatic nature of advanced non-small cell lung cancer (NSCLC), often associated with therapeutic resistance, accounts for the majority of cancer death with the 5-year survival rate less than 10%. This dismal outcome will remain the same until we gain better understanding of the crucial drivers of the metastatic process and gain the power to effectively target them. S100A4 is a tumor metastasis associated protein and an epithelial to mesenchymal transition (EMT) marker. S100A4 contributes to several hallmarks of cancer such as anti-apoptosis, proliferation and therapeutic resistance, and has been implicated in the progression of different types of cancer, including breast, colon and pancreatic cancer. However, the impact of S100A4 signaling in lung cancer progression and its potential use as a target for therapy in lung cancer has not been properly explored. Here, by using established lung cancer cell lines, we demonstrate that S100A4 is upregulated in a subset of lung cancer cell lines both at the mRNA and protein levels. We further found that inhibition of S100A4 by shRNA in A549 and H460 lung cancer cell lines reduced cell proliferation and decreased 3D invasive growth, while exogenous overexpression of S100A4 in H1299 cells increased invasive potential. Interestingly, we showed that Niclosamide, an FDA-approved anti-tapeworm agent, suppressed S100A4 expression in A549 and H358 cells and led to decreased cell proliferation, invasion and invasive growth. Similarly, we found that a CDC25 inhibitor, NSC95397, disrupts the interaction of S100A4 and non-muscle myosin IIA and also decreased A549 cell invasion. Collectively, these data highlight the importance of S100A4 in lung cancer invasion and metastasis and provide the rationale for targeting S100A4 as a potential agent to combat lung cancer. Citation Format: Min Chen, Rachel Stewart, Teresa Knifley, Brittany L. Carpenter. S100A4 drives the invasive potential of non-small cell lung cancer cells and is targeted by FDA approved drugs. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A44.

Abstract 2275: S100A4/metastasin-1 promotes lung cancer cell invasion and associates with decreased overall survival among patients with adenocarcinoma

The metastatic nature of advanced non-small cell lung cancer (NSCLC) is associated with therapeutic resistance, quick time to progression, and poor prognosis. This dismal outcome remains until we gain a better understanding of the crucial drivers of the metastatic process and the power to effectively target them. S100A4 (metastasin-1) is a tumor metastasis associated protein and an epithelial to mesenchymal transition (EMT) marker. S100A4 contributes to several hallmarks of cancer such as anti-apoptosis, proliferation, and therapeutic resistance. This protein has been implicated in the progression of different types of cancer, including breast, colon and pancreas. However, the impacts of S100A4 signaling in lung cancer progression and its potential use as a target for therapy have not been properly explored. Here, using established lung cancer cell lines, we demonstrate that S100A4 is overexpressed in about 45% of lung cancer cell lines tested, both at the mRNA and protein levels. To address the biological action of S100A4 overexpression in lung cancer cells, loss-of function and gain-of function experiments were performed. We found that inhibition of S100A4 by shRNA in A549 and H460 lung cancer cell lines reduced cell proliferation and invasion and decreased 3D invasive growth, while exogenous overexpression of S100A4 in H1299 cells increased invasive potential. In order to examine S100A4 expression in patient-derived tissues, we constructed a lung cancer tissue microarray (TMAs, N = 212) and analyzed it using immunohistochemistry. We found that S100A4 is preferentially overexpressed in lung adenocarcinoma (P = 0.0007) when compared to squamous cell carcinoma, which was confirmed using publicly available gene expression datasets (TCGA). Furthermore, we found that S100A4 overexpression is associated with the presence of lymphovascular invasion (P = 0.0189) and with decreased overall survival (Hazard ratio 2.14, 95% confidence interval 1.08-4.22, P = 0.0243) among patients with lung adenocarcinoma. To elucidate the mechanisms by which S100A4 promotes lung cancer invasive capacity, we focused on the NF-κB pathway. We observed that depletion of S100A4 by shRNA inhibited NF-κB activity and decreased TNFα-induced MMP9 expression. Furthermore, inhibition of the NF-κB/MMP9 axis decreased lung cancer cell invasive potential. Collectively, this study highlights the importance of the S100A4/NF-κB/MMP9 axis in lung cancer invasion and metastasis and provides rationale for targeting S100A4 to combat lung cancer. Citation Format: Rachel L. Stewart, Brittany L. Carpenter, Dava S. West, Teresa Knifley, Chi Wang, Heidi L. Weiss, Tamas S. Gal, Kathleen L. O9Connor, Min Chen. S100A4/metastasin-1 promotes lung cancer cell invasion and associates with decreased overall survival among patients with adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2275. doi:10.1158/1538-7445.AM2015-2275

Abstract 1943: Integrin α6β4 promotes autocrine EGFR signaling to stimulate migration and invasion toward HGF

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnIntegrin α6β4 is upregulated in pancreatic adenocarcinomas where it contributes to carcinoma cell invasion, in part, through its coordinated modulation of the transcriptome. Here, we find that integrin α6β4 signaling dramatically upregulates the expression of several genes involved in the epidermal growth factor receptor (EGFR) pathway, including amphiregulin (Areg), epiregulin (Ereg), and ectodomain cleavage protease MMP1. Notably, these genes correlate well with integrin α6β4 expression in publicly available pancreatic cancer databases. We previously reported that integrin α6β4 promotes HGF-stimulated migration and invasion; therefore, we tested the role of autocrine EGFR signaling in these processes. We discovered that blocking EGFR signaling with EGFR-specific blocking antibodies or an EGFR tyrosine kinase inhibitor hindered HGF-stimulated pancreatic carcinoma cell chemotaxis and invasive growth in three-dimensional culture. Furthermore, we found that both Areg and Ereg are required for these autocrine effects, as knocking down either ligand prevented migration and invasion. Notably, AsPC1 cells, isolated from abdominal metastases, are not dependent on EGFR signaling for migration or invasion; thus suggesting a mechanism exists for circumventing EGFR dependency. We also demonstrated that HGF stimulates the secretion of Areg, which is dependent on integrin signaling pathways including MAPK, PI3K and PKC and that Areg was transported to the leading edge of cells treated with HGF. Moreover, MMP activity and integrin α6β4 signaling are required for Areg secretion. Finally, we found that EGFR is phosphorylated in response to HGF stimulation which was dependent on EGFR protein kinase activity; however, Met phosphorylation in response to HGF was unaffected by EGFR signaling. Taken together, we show that integrin α6β4 stimulates invasion by promoting autocrine EGFR signaling through the upregulation of key genes and facilitating HGF-stimulated EGFR ligand secretion.nnCitation Format: Brittany L. Carpenter, Min Chen, Teresa Knifley, Kelley A. Davis, Susan M. Harrison, Rachel L. Stewart, Kathleen L. OConnor. Integrin α6β4 promotes autocrine EGFR signaling to stimulate migration and invasion toward HGF. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1943. doi:10.1158/1538-7445.AM2015-1943

Abstract P6-04-11: Use of synthetic isoprenoids to target protein prenylation and Rho GTPases in breast cancer invasion

Tumor metastasis causes the majority of cancer deaths. Dysregulation of small GTPases such as K-Ras and Rho family GTPases (RhoA, RhoC, Rac1 and Cdc42) is critical to drive the invasion and metastasis of a variety of cancers including breast carcinomas. In order to fulfill their biological functions, these GTPases must properly localize subcellularly and associate with cellular membrane. The association of these proteins to membranes is regulated by a series of post-translational modifications including either farnesylation or geranylgeranylation of the C-terminal CaaX motif, thus providing the rationale to target the farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) as the potential anti-cancer drugs. However, FTase inhibitors (FTIs) failed in clinic due to alternative prenylation, which causes K-Ras to be geranylgeranylated, and their inability to target geranylgeranylated proteins such as Rho family GTPases. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are synthetic farnesol or geranylgeraniol analogs that act as alternative substrates for FTase or GGTase but do not support normal prenyl function. Here, we test the ability of our lead PFIs anilinogeraniol (AGOH; farnesol analog) and anilinofarnesol (AFOH; geranylgeraniol analog) to impact the migratory and invasive capacities of breast carcinoma cells. We found that AGOH treatment effectively decreased the invasion of MDA-MBA-231 in a two-dimensional invasion assay at 100 mM while it blocked invasive growth in three-dimensional (3D) culture model in as little as 20 μM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase, which ribosylates and inactivates Rho proteins. The results demonstrate that Rho proteins are essential for the invasive growth of MDA-MB-231 cells. To determine how AGOH impacts Rho signaling, we performed RhoA and RhoC activity assays in MDA-MB-231 and MDA-MB-468 cells. We determined that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA/C activation and invasive growth where it inhibited RhoA and RhoC activation completely at 20 μM and blocked the invasive growth of MDA-MB-231 cells at ≤ 5 μM. Interestingly, both AGOH and AFOH had no effect on the growth of MCF10A cells in 3D culture. Collectively, this study demonstrates that our lead PFI compounds dramatically inhibited breast cancer invasion, at least in part, through affecting RhoA and RhoC function; thus, suggesting that targeting the function of Rho prenylation by PFIs will offer a promising mechanism for the treatment of breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-11.

Abstract 1647: Integrin α6β4 stimulates transcription and secretion of EGFR ligands.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnPancreatic carcinoma has the highest death to incidence ratio of all cancer due to frequent late diagnosis, drug-resistance and high metastatic potential. The integrin α6β4 is overexpressed in pancreatic carcinoma and plays an important role in tumor invasion and metastasis, in part by altering the transcriptome toward an invasive phenotype. In this study, we find that these transcriptional changes in pancreatic carcinomas include upregulation of cancer promoting genes such as ligands of the epidermal growth factor receptor (EGFR), epiregulin (Ereg) and amphiregulin (Areg). We have found previously that integrin α6β4 alters transcription by selectively targeting DNA demethylation or transcription factors, such as NFAT1 and NFAT5, to gene promoters. Given that Areg and Ereg are upregulated by over 1000-fold, we hypothesized that integrin α6β4 may control their expression by targeting their promoters for DNA demethylation. To test this concept, pancreatic cancer cells with low expression of integrin α6β4 were treated with DNA methyltransferase inhibitor 5-aza-2’deoxycytidine (DAC), and mRNA expression measured by Q-PCR. This treatment resulted in induction of Areg and Ereg expression indicating that these genes may be controlled by DNA methylation. Furthermore, suppression of integrin α6β4 by RNAi, hindered induction by DAC, thus suggesting that integrin α6β4 plays a role in ligand expression. To support these results, pancreatic cancer cells were treated with the methyl donor S-adenosylmethionine, allowing for global hypermethylation and silencing of genes controlled by methylation. Our results show that treatment inhibits Ereg and Areg expression in cells with high expression of integrin α6β4, providing evidence for epigenetic regulation by this integrin. Non-coding RNAs processed by Dicer often play a role in DNA methylation of specific genes. Accordingly, we used siRNA to target the microRNA processing enzyme Dicer. Dicer knockdown resulted in decreased expression of Ereg and Areg, measured by Q-PCR, indicating that small non-coding RNAs are involved in their regulation. Autocrine EGFR signaling is also regulated by altered secretion of Ereg and Areg. Using an ELISA for Areg, we measured secretion associated with variable expression of integrin α6β4. We find that integrin α6β4 stimulated secretion of Areg into the media, and this secretion is enhanced by HGF signaling. Consequently, we also provide evidence that integrin α6β4 promotes HGF-stimulated chemotaxis, invasion, and invasive growth which is facilitated by autocrine EGFR signaling. This study demonstrates that overexpression of integrin α6β4 in pancreatic cancer promotes a more aggressive phenotype by activating transcriptional expression and autocrine secretion of EGFR ligands, Ereg and Areg, that promote an invasive phenotype.nnCitation Format: Brittany L. Carpenter, Min Chen, Teresa Knifley, Kathleen L. OConnor. Integrin α6β4 stimulates transcription and secretion of EGFR ligands. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1647. doi:10.1158/1538-7445.AM2013-1647

Abstract 2349: Integrin α6β4 contributes to HGF-stimulated migration and invasion by promoting autocrine EGFR signaling in pancreatic cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnThe lethality of pancreatic cancer is due to elevated incidences of invasion and metastasis. Previous studies from our lab and others show that pro-invasive integrin α6β4 is upregulated in early stage pancreatic cancer, maintained during tumor progression and, in turn, promotes chemotactic migration and invasion toward HGF. To gain insight into the mechanisms governing how integrin α6β4 promotes these processes, we performed Affymetrix Gene Chip analysis on Panc-1 clones with differing levels of integrin α6β4 expression. After statistical processing, we found 582 genes altered by integrin α6β4 signaling. From these results, we found that high integrin α6β4-expressing Panc1 clones substantially enhanced expression of genes affecting epidermal growth factor receptor (EGFR) signaling. The genes upregulated by integrin α6β4 included EGFR, EGF-like ligands amphiregulin (AREG) and epiregulin (EREG), ectodomain cleavage proteases ADAMTS1, MMP1, and hyaluronan synthase 3. The upregulation of these genes was confirmed by real-time PCR, immunoblot analysis and/or ELISA. Stable overexpression of integrin β4 in low integrin β4 expressing cells increased the levels of AREG and EREG by real-time PCR. We further tested the role of autocrine EGFR signaling by pretreating cells with EGFR blocking antibodies (LA1) or EGFR-specific protein tyrosine kinase inhibitor (PD153035) and tested HGF-stimulated chemotaxis, chemo-invasion and invasive growth in three-dimensional culture. Interestingly, these inhibitors blocked integrin α6β4-dependent HGF-stimulated chemotaxis, invasion and invasive growth. In summary, our data suggest integrin α6β4 confers a motile and invasive phenotype to pancreatic carcinoma cells, in part, by coordinately regulating expression of key elements of autocrine EGFR signaling.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2349. doi:10.1158/1538-7445.AM2011-2349