FAK, vinculin, and talin control mechanosensitive YAP nuclear localization.

Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity.

Carnosic Acid against Lung Cancer: Induction of Autophagy and Activation of Sestrin-2/LKB1/AMPK Signalling.

Non-small cell lung cancer (NSCLC) represents 80% of all lung cancer cases and is characterized by low survival rates due to chemotherapy and radiation resistance. Novel treatment strategies for NSCLC are urgently needed. Liver kinase B1 (LKB1), a tumor suppressor prevalently mutated in NSCLC, activates AMP-activated protein kinase (AMPK) which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) and activates unc-51 like autophagy activating kinase 1 (ULK1) to promote autophagy. Sestrin-2 is a stress-induced protein that enhances LKB1-dependent activation of AMPK, functioning as a tumor suppressor in NSCLC. In previous studies, rosemary (Rosmarinus officinalis) extract (RE) activated the AMPK pathway while inhibiting mTORC1 to suppress proliferation, survival, and migration, leading to the apoptosis of NSCLC cells. In the present study, we investigated the anticancer potential of carnosic acid (CA), a bioactive polyphenolic diterpene compound found in RE. The treatment of H1299 and H460 NSCLC cells with CA resulted in concentration and time-dependent inhibition of cell proliferation assessed with crystal violet staining and 3H-thymidine incorporation, and concentration-dependent inhibition of survival, assessed using a colony formation assay. Additionally, CA induced apoptosis of H1299 cells as indicated by decreased B-cell lymphoma 2 (Bcl-2) levels, increased cleaved caspase-3, -7, poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (BAX) levels, and increased nuclear condensation. These antiproliferative and proapoptotic effects coincided with the upregulation of sestrin-2 and the phosphorylation/activation of LKB1 and AMPK. Downstream of AMPK signaling, CA increased levels of autophagy marker light chain 3 (LC3), an established marker of autophagy; inhibiting autophagy with 3-methyladenine (3MA) blocked the antiproliferative effect of CA. Overall, these data indicate that CA can inhibit NSCLC cell viability and that the underlying mechanism of action of CA involves the induction of autophagy through a Sestrin-2/LKB1/AMPK signaling cascade. Future experiments will use siRNA and small molecule inhibitors to better elucidate the role of these signaling molecules in the mechanism of action of CA as well as tumor xenograft models to assess the anticancer properties of CA in vivo.

Integrative modeling and analysis of signaling crosstalk reveal molecular switches coordinating Yes-associated protein transcriptional activities.

The transcriptional co-activator YAP forms complexes with distinct transcription factors, controlling cell fate decisions, such as proliferation and apoptosis. However, the mechanisms underlying its context-dependent function are poorly defined. This study explores the interplay between the TGF-ß and Hippo pathways and their influence on YAP's association with specific transcription factors. By integrating iterative mathematical modeling with experimental validation, we uncover molecular switches, predominantly controlled by RASSF1A and ITCH, which dictate the formation of YAP-SMAD (proliferative) and YAP-p73 (apoptotic) complexes. Our results show that RASSF1A enhances the formation of apoptotic complexes, whereas ITCH promotes the formation of proliferative complexes. Notably, higher levels of ITCH transform YAP-SMAD activity from a transient to a sustained state, impacting cellular behaviors. Extending these findings to various breast cancer cell lines highlights the role of cellular context in YAP regulation. Our study provides new insights into the mechanisms of YAP transcriptional activities and their therapeutic implications.

Targeted irradiation in an autochthonous mouse model of pancreatic cancer.

The value of radiotherapy in the treatment of pancreatic cancer has been the subject of much debate but limited preclinical research. We hypothesise that the poor translation of radiation research into clinical trials of radiotherapy in pancreatic cancer is due, in part, to inadequate preclinical study models. Here, we developed and refined methods for targeted irradiation in autochthonous mouse models of pancreatic cancer, using a small animal radiotherapy research platform. We tested and optimised strategies for administration of contrast agents, iohexol and the liver imaging agent Fenestra LC, to enable the use of computed tomography imaging in tumour localisation. We demonstrate accurate tumour targeting, negligible off-target effects and therapeutic efficacy, depending on dose, number of fractions and tumour size, and provide a proof of concept that precise radiation can be delivered effectively to mouse pancreatic tumours with a clinically relevant microenvironment. This advance will allow investigation of the radiation response in murine pancreatic cancer, discovery of mechanisms and biomarkers of radiosensitivity or resistance, and development of radiosensitising strategies to inform clinical trials for precision radiotherapy in this disease.

p53 inhibitor iASPP is an unexpected suppressor of KRAS and inflammation-driven pancreatic cancer.

Oncogenic KRAS activation, inflammation and p53 mutation are key drivers of pancreatic cancer (PC) development. Here we report iASPP, an inhibitor of p53, as a paradoxical suppressor of inflammation and oncogenic KRASG12D-driven PC tumorigenesis. iASPP suppresses PC onset driven by KRASG12D alone or KRASG12D in combination with mutant p53R172H. iASPP deletion limits acinar-to-ductal metaplasia (ADM) in vitro but accelerates inflammation and KRASG12D-induced ADM, pancreatitis and PC tumorigenesis in vivo. KRASG12D/iASPPΔ8/Δ8 tumours are well-differentiated classical PCs and their derivative cell lines form subcutaneous tumours in syngeneic and nude mice. Transcriptomically, either iASPP deletion or p53 mutation in the KRASG12D background altered the expression of an extensively overlapping gene set, comprised primarily of NF-κB and AP1-regulated inflammatory genes. All these identify iASPP as a suppressor of inflammation and a p53-independent oncosuppressor of PC tumorigenesis.

Effects of Berberine against Pancreatitis and Pancreatic Cancer.

The pancreas is a glandular organ with endocrine and exocrine functions necessary for the maintenance of blood glucose homeostasis and secretion of digestive enzymes. Pancreatitis is characterized by inflammation of the pancreas leading to temporary or permanent pancreatic dysfunction. Inflammation and fibrosis caused by chronic pancreatitis exacerbate malignant transformation and significantly increase the risk of developing pancreatic cancer, the world's most aggressive cancer with a 5-year survival rate less than 10%. Berberine (BBR) is a naturally occurring plant-derived polyphenol present in a variety of herbal remedies used in traditional medicine to treat ulcers, infections, jaundice, and inflammation. The current review summarizes the existing in vitro and in vivo evidence on the effects of BBR against pancreatitis and pancreatic cancer with a focus on the signalling mechanisms underlying the effects of BBR.

53BP1-mediated recruitment of RASSF1A to ribosomal DNA breaks promotes local ATM signaling.

DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a relatively greater impact on overall genome stability. The ribosomal RNA gene repeats (rDNA) are emerging fragile sites. Recent progress in understanding how the rDNA damage response is organized has highlighted a key role of adaptor proteins. Here, we show that the scaffold tumor suppressor RASSF1A is recruited to rDNA breaks. RASSF1A recruitment to double-strand breaks is mediated by 53BP1 and depends on RASSF1A phosphorylation at Serine 131 by ATM kinase. Employing targeted rDNA damage, we uncover that RASSF1A recruitment promotes local ATM signaling. RASSF1A silencing, a common epigenetic event during malignant transformation, results in persistent breaks, rDNA copy number alterations and decreased cell viability. Overall, we identify a novel role for RASSF1A at rDNA break sites, provide mechanistic insight into how the DNA damage response is organized in a chromatin context, and provide further evidence for how silencing of the RASSF1A tumor suppressor contributes to genome instability.

Chemical-induced gene expression ranking and its application to pancreatic cancer drug repurposing.

Chemical-induced gene expression profiles provide critical information of chemicals in a biological system, thus offering new opportunities for drug discovery. Despite their success, large-scale analysis leveraging gene expressions is limited by time and cost. Although several methods for predicting gene expressions were proposed, they only focused on imputation and classification settings, which have limited applications to real-world scenarios of drug discovery. Therefore, a chemical-induced gene expression ranking (CIGER) framework is proposed to target a more realistic but more challenging setting in which overall rankings in gene expression profiles induced by de novo chemicals are predicted. The experimental results show that CIGER significantly outperforms existing methods in both ranking and classification metrics. Furthermore, a drug screening pipeline based on CIGER is proposed to identify potential treatments of drug-resistant pancreatic cancer. Our predictions have been validated by experiments, thereby showing the effectiveness of CIGER for phenotypic compound screening of precision medicine.

Predicting Early Disease Recurrence of Pancreatic Cancer following Surgery: Determining the Role of NUDT15 as a Prognostic Biomarker.

Surgical resection remains the only curative treatment strategy for Pancreatic Ductal Adenocarcinoma (PDAC). A proportion of patients succumb to early disease recurrence post-operatively despite receiving adjuvant chemotherapy. The ability to identify these high-risk individuals at their initial diagnosis, prior to surgery, could potentially alter their treatment algorithm. This unique patient cohort may benefit from neo-adjuvant chemotherapy, even in the context of resectable disease, as this may secure systemic control over their disease burden. It may also improve patient selection for surgery. Using the Cancer Genome Atlas dataset, we first confirmed the poor overall survival associated with early disease recurrence (p < 0.0001). The transcriptomic profiles of these tumours were analysed, and we identified key aberrant signalling pathways involved in early disease relapse; downregulation across several immune signalling pathways was noted. Differentially expressed genes that could serve as biomarkers were identified (BPI, C6orf58, CD177, MCM7 and NUDT15). Receiver operating characteristic curves were constructed in order to identify biomarkers with a high diagnostic ability to identify patients who developed early disease recurrence. NUDT15 expression had the highest discriminatory capability as a biomarker (AUC 80.8%). Its expression was confirmed and validated in an independent cohort of patients with resected PDAC (n = 13). Patients who developed an early recurrence had a statistically higher tumour expression of NUDT15 when compared to patients who did not recur early (p < 0.01). Our results suggest that NUDT15 can be used as a prognostic biomarker that can stratify patients according to their risk of developing early disease recurrence.

Circulating proteins and risk of pancreatic cancer: a case-subcohort study among Chinese adults.

BACKGROUND: Pancreatic cancer has a very poor prognosis. Biomarkers that may help predict or diagnose pancreatic cancer may lead to earlier diagnosis and improved survival. METHODS: The prospective China Kadoorie Biobank (CKB) recruited 512 891 adults aged 30-79 years during 2004-08, recording 702 incident cases of pancreatic cancer during 9 years of follow-up. We conducted a case-subcohort study measuring 92 proteins in 610 cases and a subcohort of 623 individuals, using the OLINK immuno-oncology panel in stored baseline plasma samples. Cox regression with the Prentice pseudo-partial likelihood was used to estimate adjusted hazard ratios (HRs) for risk of pancreatic cancer by protein levels. RESULTS: Among 1233 individuals (including 610 cases), several chemokines, interleukins, growth factors and membrane proteins were associated with risk of pancreatic cancer, with adjusted HRs per 1 standard deviation (SD) of 0.86 to 1.86, including monocyte chemotactic protein 3 (MCP3/CCL7) {1.29 [95% CI (confidence interval) (1.10, 1.51)]}, angiopoietin-2 (ANGPT2) [1.27 (1.10, 1.48)], interleukin-18 (IL18) [1.24 (1.07, 1.43)] and interleukin-6 (IL6) [1.21 (1.06, 1.38)]. Associations between some proteins [e.g. matrix metalloproteinase-7 (MMP7), hepatocyte growth factor (HGF) and tumour necrosis factor receptor superfamily member 9 [TNFRSF9)] and risk of pancreatic cancer were time-varying, with higher levels associated with higher short-term risk. Within the first year, the discriminatory ability of a model with known risk factors (age, age squared, sex, region, smoking, alcohol, education, diabetes and family history of cancer) was increased when several proteins were incorporated (weighted C-statistic changed from 0.85 to 0.99; P for difference = 4.5 × 10-5), although only a small increase in discrimination (0.77 to 0.79, P = 0.04) was achieved for long-term risk. CONCLUSIONS: Several plasma proteins were associated with subsequent diagnosis of pancreatic cancer. The potential clinical utility of these biomarkers warrants further investigation.

RASSF1A disrupts the NOTCH signaling axis via SNURF/RNF4-mediated ubiquitination of HES1.

RASSF1A promoter methylation has been correlated with tumor dedifferentiation and aggressive oncogenic behavior. Nevertheless, the underlying mechanism of RASSF1A-dependent tumor dedifferentiation remains elusive. Here, we show that RASSF1A directly uncouples the NOTCH-HES1 axis, a key suppressor of differentiation. Interestingly, the crosstalk of RASSF1A with HES1 occurs independently from the signaling route connecting RASSF1A with the Hippo pathway. At the molecular level, we demonstrate that RASSF1A acts as a scaffold essential for the SUMO-targeted E3 ligase SNURF/RNF4 to target HES1 for degradation. The reciprocal relationship between RASSF1A and HES1 is evident across a wide range of human tumors, highlighting the clinical significance of the identified pathway. We show that HES1 upregulation in a RASSF1A-depleted environment renders cells non-responsive to the downstream effects of γ-secretase inhibitors (GSIs) which restrict signaling at the level of the NOTCH receptor. Taken together, we report a mechanism through which RASSF1A exerts autonomous regulation of the critical Notch effector HES1, thus classifying RASSF1A expression as an integral determinant of the clinical effectiveness of Notch inhibitors.

Novel Galectin-3 Roles in Neurogenesis, Inflammation and Neurological Diseases.

Galectin-3 (Gal-3) is an evolutionarily conserved and multifunctional protein that drives inflammation in disease. Gal-3's role in the central nervous system has been less studied than in the immune system. However, recent studies show it exacerbates Alzheimer's disease and is upregulated in a large variety of brain injuries, while loss of Gal-3 function can diminish symptoms of neurodegenerative diseases such as Alzheimer's. Several novel molecular pathways for Gal-3 were recently uncovered. It is a natural ligand for TREM2 (triggering receptor expressed on myeloid cells), TLR4 (Toll-like receptor 4), and IR (insulin receptor). Gal-3 regulates a number of pathways including stimulation of bone morphogenetic protein (BMP) signaling and modulating Wnt signalling in a context-dependent manner. Gal-3 typically acts in pathology but is now known to affect subventricular zone (SVZ) neurogenesis and gliogenesis in the healthy brain. Despite its myriad interactors, Gal-3 has surprisingly specific and important functions in regulating SVZ neurogenesis in disease. Gal-1, a similar lectin often co-expressed with Gal-3, also has profound effects on brain pathology and adult neurogenesis. Remarkably, Gal-3's carbohydrate recognition domain bears structural similarity to the SARS-CoV-2 virus spike protein necessary for cell entry. Gal-3 can be targeted pharmacologically and is a valid target for several diseases involving brain inflammation. The wealth of molecular pathways now known further suggest its modulation could be therapeutically useful.

RASSF1C oncogene elicits amoeboid invasion, cancer stemness, and extracellular vesicle release via a SRC/Rho axis.

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal-to-amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC-mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C-induced amoeboid cells display increased expression of cancer stem-like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C-induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C-driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early-stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.

Dynamic Physiological Culture of Ex Vivo Human Tissue: A Systematic Review.

Conventional static culture fails to replicate the physiological conditions that exist in vivo. Recent advances in biomedical engineering have resulted in the creation of novel dynamic culturing systems that permit the recapitulation of normal physiological processes ex vivo. Whilst the physiological benefit for its use in the culture of two-dimensional cellular monolayer has been validated, its role in the context of primary human tissue culture has yet to be determined. This systematic review identified 22 articles that combined dynamic physiological culture techniques with primary human tissue culture. The most frequent method described (55%) utilised dynamic perfusion culture. A diverse range of primary human tissue was successfully cultured. The median duration of successful ex vivo culture of primary human tissue for all articles was eight days; however, a wide range was noted (5 h-60 days). Six articles (27%) reported successful culture of primary human tissue for greater than 20 days. This review illustrates the physiological benefit of combining dynamic culture with primary human tissue culture in both long-term culture success rates and preservation of native functionality of the tissue ex vivo. Further research efforts should focus on developing precise biochemical sensors that would allow for real-time monitoring and automated self-regulation of the culture system in order to maintain homeostasis. Combining these techniques allows the creation of an accurate system that can be used to gain a greater understanding of human physiology.

Force-FAK signaling coupling at individual focal adhesions coordinates mechanosensing and microtissue repair.

How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization as well as traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair.

TET2 Drives 5hmc Marking of GATA6 and Epigenetically Defines Pancreatic Ductal Adenocarcinoma Transcriptional Subtypes.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by advanced disease stage at presentation, aggressive disease biology, and resistance to therapy, resulting in an extremely poor 5-year survival rate of <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression, but exactly why is unclear and is hindered by the technical limitations of analyzing clinical samples. METHODS: We performed genome-wide epigenetic mapping of DNA modifications 5-methylcytosine and 5-hydroxymethylcytosine (5hmc) using oxidative bisulfite sequencing from formalin-embedded sections. We identified overlap with transcriptional signatures in formalin-fixed, paraffin-embedded tissue from resected patients, via bioinformatics using iCluster and mutational profiling and confirmed them in vivo. RESULTS: We found that aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci, including GATA6, which promote differentiated classical pancreatic subtypes. We showed that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5-methylcytosine hydroxylase TET2. Furthermore, we found that SMAD4 directly supports TET2 levels in classical pancreatic tumors, and loss of SMAD4 expression was associated with reduced 5hmc, GATA6, and squamous-like tumors. Importantly, enhancing TET2 stability using metformin and vitamin C/ascorbic acid restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo. CONCLUSIONS: We identified epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data showed that restoring epigenetic control increases biomarkers of classical pancreatic tumors that are associated with improved therapeutic responses and survival.

Geography is more important than life history in the recent diversification of the tiger salamander complex.

The North American tiger salamander species complex, including its best-known species, the Mexican axolotl, has long been a source of biological fascination. The complex exhibits a wide range of variation in developmental life history strategies, including populations and individuals that undergo metamorphosis; those able to forego metamorphosis and retain a larval, aquatic lifestyle (i.e., paedomorphosis); and those that do both. The evolution of a paedomorphic life history state is thought to lead to increased population genetic differentiation and ultimately reproductive isolation and speciation, but the degree to which it has shaped population- and species-level divergence is poorly understood. Using a large multilocus dataset from hundreds of samples across North America, we identified genetic clusters across the geographic range of the tiger salamander complex. These clusters often contain a mixture of paedomorphic and metamorphic taxa, indicating that geographic isolation has played a larger role in lineage divergence than paedomorphosis in this system. This conclusion is bolstered by geography-informed analyses indicating no effect of life history strategy on population genetic differentiation and by model-based population genetic analyses demonstrating gene flow between adjacent metamorphic and paedomorphic populations. This fine-scale genetic perspective on life history variation establishes a framework for understanding how plasticity, local adaptation, and gene flow contribute to lineage divergence. Many members of the tiger salamander complex are endangered, and the Mexican axolotl is an important model system in regenerative and biomedical research. Our results chart a course for more informed use of these taxa in experimental, ecological, and conservation research.

Anti-Cancer Properties of Theaflavins.

Cancer is a disease characterized by aberrant proliferative and apoptotic signaling pathways, leading to uncontrolled proliferation of cancer cells combined with enhanced survival and evasion of cell death. Current treatment strategies are sometimes ineffective in eradicating more aggressive, metastatic forms of cancer, indicating the need to develop novel therapeutics targeting signaling pathways which are essential for cancer progression. Historically, plant-derived compounds have been utilized in the production of pharmaceuticals and chemotherapeutic compounds for the treatment of cancer, including paclitaxel and docetaxel. Theaflavins, phenolic components present in black tea, have demonstrated anti-cancer potential in cell cultures in vitro and in animal studies in vivo. Theaflavins have been shown to inhibit proliferation, survival, and migration of many cancer cellswhile promoting apoptosis. Treatment with theaflavins has been associated with increased levels of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspases-3, -7, -8, and -9, all markers of apoptosis, and increased expression of the proapoptotic marker Bcl-2-associated X protein (Bax) and concomitant reduction in the antiapoptotic marker B-cell lymphoma 2 (Bcl-2). Additionally, theaflavin treatment reduced phosphorylated Akt, phosphorylated mechanistic target of rapamycin (mTOR), phosphatidylinositol 3-kinase (PI3K), and c-Myc levels with increased expression of the tumour suppressor p53. This review summarizes the current in vitro and in vivo evidence available investigating the anti-cancer effects of theaflavins across various cancer cell lines and animal models.

Inhibition of Non-Small Cell Lung Cancer Proliferation and Survival by Rosemary Extract Is Associated with Activation of ERK and AMPK.

Non-small cell lung cancer (NSCLC) represents an aggressive form of lung cancer which often develops resistance to chemo- and radiotherapy emphasizing a need to identify novel treatment agents to combat it. Many plants contain compounds with anti-inflammatory, antimicrobial, antidiabetic, and anticancer properties and some plant-derived chemicals are used in the treatment of cancer. A limited number of in vitro and in vivo animal studies provide evidence of anticancer effects of rosemary (Rosmarinus officinalis) extract (RE); however, no studies have explored its role in H1299 NSCLC cells, and its underlying mechanism(s) of action are not understood. The current study examined the effects of RE on H1299 cell proliferation, survival, and migration using specific assays. Additionally, immunoblotting was used to investigate the effects of RE treatment on signalling molecules implicated in cell growth and survival. Treatment with RE dose-dependently inhibited H1299 proliferation with an IC50 value of 19 µg/mL. Similarly, RE dose-dependently reduced cell survival, and this reduction correlated with increased levels of cleaved poly (ADP-ribose) polymerase (PARP), a marker of apoptosis. RE was also able to inhibit cell migration as assessed with a wound healing assay. These cellular effects of RE were associated with an increase in phosphorylated levels of extracellular signal-regulated kinase (ERK), AMP-activated protein kinase (AMPK), and its downstream targets ACC, the mTORC1 protein raptor, and decreased p70S6K phosphorylation. More studies are required to fully examine the effects of RE against NSCLC.