Integrin α6ß4 signals through DNA damage response pathway to sensitize breast cancer cells to cisplatin.

Integrin α6ß4 is highly expressed in triple negative breast cancer (TNBC) and drives its most aggressive traits; however, its impact on chemotherapeutic efficacy remains untested. We found that integrin α6ß4 signaling promoted sensitivity to cisplatin and carboplatin but not to other chemotherapies tested. Mechanistic investigations revealed that integrin α6ß4 stimulated the activation of ATM, p53, and 53BP1, which required the integrin ß4 signaling domain. Genetic manipulation of gene expression demonstrated that mutant p53 cooperated with integrin α6ß4 for cisplatin sensitivity and was necessary for downstream phosphorylation of 53BP1 and enhanced ATM activation. Additionally, we found that in response to cisplatin-induced DNA double strand break (DSB), integrin α6ß4 suppressed the homologous recombination (HR) activity and enhanced non-homologous end joining (NHEJ) repair activity. Finally, we discovered that integrin α6ß4 preferentially activated DNA-PK, facilitated DNA-PK-p53 and p53-53BP1 complex formation in response to cisplatin and required DNA-PK to enhance ATM, 53BP1 and p53 activation as well as cisplatin sensitivity. In summary, we discovered a novel function of integrin α6ß4 in promoting cisplatin sensitivity in TNBC through DNA damage response pathway.

Integrin α6ß4 requires plectin and vimentin for adhesion complex distribution and invasive growth.

Integrin α6ß4 binds plectin to associate with vimentin; however, the biological function remains unclear. Here, we utilized various integrin ß4 mutants and CRISPR-Cas9 editing to investigate this association. Upon laminin binding, integrin α6ß4 distinctly distributed peripherally as well as centrally, proximal to the nucleus. Upon fibronectin addition, integrin α6ß4 was centrally recruited to large focal adhesions (FAs) and enhanced Fak (also known as PTK2) phosphorylation. Integrin ß4 plectin-binding mutants or genetic deletion of plectin inhibited ß4 recruitment to FAs and integrin α6ß4-enhanced cell spreading, migration and three-dimensional invasive growth. Loss of the ß4 signaling domain (but retaining plectin binding) blocked migration and invasiveness but not cell spreading, recruitment to FAs or colony growth. Immunostaining revealed that integrin α6ß4 redistributed vimentin perinuclearly, where it colocalized with plectin and FAs. Depletion of vimentin completely blocked integrin ß4-enhanced invasive growth, Fak phosphorylation and proliferation in three dimensions but not two dimensions. In summary, we demonstrate the essential roles of plectin and vimentin in promoting an invasive phenotype downstream of integrin α6ß4. This article has an associated First Person interview with the first author of the paper.

In vivo Tumor Growth and Spontaneous Metastasis Assays Using A549 Lung Cancer Cells.

Metastasis accounts for the majority of cancer related deaths. The genetically engineered mouse (GEM) models and cell line-based subcutaneous and orthotopic mouse xenografts have been developed to study the metastatic process. By using lung cancer cell line A549 as an example, we present a modified protocol to establish the cell line-based xenograft. Our protocol ensures sufficient establishment of the mouse xenografts and allows us to monitor tumor growth and spontaneous metastasis. This protocol could be adapted to other types of established cancer cell lines or primary cancer cells to study the mechanism of metastatic process as well as to test the effect of the potential anti-cancer agents on tumor growth and metastatic capacity.

S100A4 alters metabolism and promotes invasion of lung cancer cells by up-regulating mitochondrial complex I protein NDUFS2.

It is generally accepted that alterations in metabolism are critical for the metastatic process; however, the mechanisms by which these metabolic changes are controlled by the major drivers of the metastatic process remain elusive. Here, we found that S100 calcium-binding protein A4 (S100A4), a major metastasis-promoting protein, confers metabolic plasticity to drive tumor invasion and metastasis of non-small cell lung cancer cells. Investigating how S100A4 regulates metabolism, we found that S100A4 depletion decreases oxygen consumption rates, mitochondrial activity, and ATP production and also shifts cell metabolism to higher glycolytic activity. We further identified that the 49-kDa mitochondrial complex I subunit NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NDUFS2) is regulated in an S100A4-dependent manner and that S100A4 and NDUFS2 exhibit co-occurrence at significant levels in various cancer types as determined by database-driven analysis of genomes in clinical samples using cBioPortal for Cancer Genomics. Importantly, we noted that S100A4 or NDUFS2 silencing inhibits mitochondrial complex I activity, reduces cellular ATP level, decreases invasive capacity in three-dimensional growth, and dramatically decreases metastasis rates as well as tumor growth in vivo Finally, we provide evidence that cells depleted in S100A4 or NDUFS2 shift their metabolism toward glycolysis by up-regulating hexokinase expression and that suppressing S100A4 signaling sensitizes lung cancer cells to glycolysis inhibition. Our findings uncover a novel S100A4 function and highlight its importance in controlling NDUFS2 expression to regulate the plasticity of mitochondrial metabolism and thereby promote the invasive and metastatic capacity in lung cancer.

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 α6ß4 Promotes Autocrine Epidermal Growth Factor Receptor (EGFR) Signaling to Stimulate Migration and Invasion toward Hepatocyte Growth Factor (HGF).

Integrin α6ß4 is up-regulated in pancreatic adenocarcinomas where it contributes to carcinoma cell invasion by altering the transcriptome. In this study, we found that integrin α6ß4 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 α6ß4 was confirmed in The Cancer Genome Atlas Pancreatic Cancer Database. Based on previous observations that integrin α6ß4 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 α6ß4 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 α6ß4 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.

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

BACKGROUND: Various 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. METHODS: To 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. RESULTS: LPA, 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. CONCLUSIONS: These 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.