Cell Anchorage-independent Growth Research Articles

The EGFR-STYK1-FGF1 axis sustains functional drug tolerance to EGFR inhibitors in EGFR-mutant non-small cell lung cancer

Non-small cell lung cancer (NSCLC) patients harboring activating mutations in epidermal growth factor receptor (EGFR) are sensitive to therapy with EGFR tyrosine kinase inhibitors (TKI). Despite remarkable clinical responses using EGFR TKI, surviving drug tolerant cells serve as a reservoir from which drug resistant tumors may emerge. This study addresses the need for improved efficacy of EGFR TKI by identifying targets involved in functional drug tolerance against them. To this aim, a high-throughput siRNA kinome screen was performed using two EGFR TKI-sensitive EGFR-mutant NSCLC cell lines in the presence/absence of the second-generation EGFR TKI afatinib. From the screen, Serine/Threonine/Tyrosine Kinase 1 (STYK1) was identified as a target that when downregulated potentiates the effects of EGFR inhibition in vitro. We found that chemical inhibition of EGFR combined with the siRNA-mediated knockdown of STYK1 led to a significant decrease in cancer cell viability and anchorage-independent cell growth. Further, we show that STYK1 selectively interacts with mutant EGFR and that the interaction is disrupted upon EGFR inhibition. Finally, we identified fibroblast growth factor 1 (FGF1) as a downstream effector of STYK1 in NSCLC cells. Accordingly, downregulation of STYK1 counteracted the afatinib-induced upregulation of FGF1. Altogether, we unveil STYK1 as a valuable target to repress the pool of surviving drug tolerant cells arising upon EGFR inhibition. Co-targeting of EGFR and STYK1 could lead to a better overall outcome for NSCLC patients.

Cystathionine-β-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation.

Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display upregulated cystathionine-β-synthase (CBS) expression and enhanced uptake of exogenous cysteine, which lead to increased hydrogen sulfide (H2S) and glutathione (GSH) production, consequently protecting senescent cells from oxidative stress-induced cell death. CBS depletion allows AIS cells to escape senescence and re-enter the cell cycle, indicating the importance of CBS activity in maintaining AIS. Mechanistically, we show this restoration of proliferation is mediated through suppressing mitochondrial respiration and reactive oxygen species (ROS) production by reducing mitochondrial localized CBS while retaining antioxidant capacity of transsulfuration pathway. These findings implicate a potential tumor-suppressive role for CBS in cells with aberrant PI3K/AKT pathway activation. Consistent with this concept, in human gastric cancer cells with activated PI3K/AKT signaling, we demonstrate that CBS expression is suppressed due to promoter hypermethylation. CBS loss cooperates with activated PI3K/AKT signaling in promoting anchorage-independent growth of gastric epithelial cells, while CBS restoration suppresses the growth of gastric tumors in vivo. Taken together, we find that CBS is a novel regulator of AIS and a potential tumor suppressor in PI3K/AKT-driven gastric cancers, providing a new exploitable metabolic vulnerability in these cancers.

Ras suppressor-1 (RSU1) exerts a tumor suppressive role with prognostic significance in lung adenocarcinoma.

Ras suppressor-1 (RSU1), originally described as a suppressor of Ras oncogenic transformation, localizes to focal adhesions interacting with the ILK-PINCH-PARVIN (IPP) complex that exerts a well-established oncogenic role in cancer. However, RSU1 implication in lung cancer is currently unknown. Our study aims to address the role of RSU1 in lung adenocarcinoma (LUADC). We here show that RSU1 protein expression by immunohistochemistry is downregulated in LUADC human tissue samples and represents a significant prognostic indicator. In silico analysis of gene chip and RNA seq data validated our findings. Depletion of RSU1 by siRNA in lung cancer cells promotes anchorage-independent cell growth, cell motility and epithelial to mesenchymal transition (EMT). Silencing of RSU1 also alters IPP complex expression in lung cancer cells. The p29 RSU1 truncated isoform is detected in lung cancer cells, and its expression is downregulated upon RSU1 silencing, whereas it is overexpressed upon ILK overexpression. These findings suggest that RSU1 exerts a tumor suppressive role with prognostic significance in LUADC.

Abstract 1582: Chloroquine inhibites Rab11 mediated E-cadherin transport in collective colon cancer cell migration

Abstract Cell migration is regulated by multiple molecular pathways to change the morphology from epithelial to mesenchymal type and is important in cancer metastasis. In has been demonstrated epithelial mesenchymal transition (EMT) is related to E-cadherin expression level. The loss of E-cadherin expression results in tumor proliferation and metastasis. Recently, studies have been demonstrated the collective cancer cell migration which expresses high E-cadherin is a powerful mechanism of metastasis. Therefore, the role of E-cadherin in cancer metastasis is still not clear. Clathrin-mediated endocytosis (CME) was shown to regulate E-cadherin turnover on the membrane and the function of lysosome is also associated with cell vesicle trafficking, however, the role of lysosome in collective cell migration is not known. This study is investigating the role of lysosome in regulating E-cadherin dynamics through vesicle trafficking in collective colon cell migration. In the experiment, we used colon cancer cells with epithelial morphology and observed the relation of E-cadherin with endocytic protein clathrin, Rab5 and the recycling protein Rab11 in collective migrated cells. In the result, inhibition of lysosome activity by chloroquine treatment has great suppression effect on anchorage independent cell growth and inhibited collective cell migration. We observed that nocodazole treatment inhibited clathrin-mediated endocytosis, which caused E-cadherin to accumulate on the cell membrane. Western blot and confocal microscopy demonstrated chloroquine treatment induced Rab11 expression and accumulated in the cytosol which affected E-cadherin turnover. Our results demonstrated inhibits lysosome activity can suppress colon cancer cell growth and collective migration through lysosome mediated E-cadherin translocation. Chloroquine may be the potential treatment for colon cancer therapy. keywords: colon cancer, collective cell migration, E-cadherin, vesicle trafficking, lysosome Citation Format: Xiang-Ling Hou, Hui Min Koo, Yu-Cheng Weng, Yi-Wen Lu, Viriya Adhiguna Winarso, Yu-Lin Li, Wei-Ting Chao. Chloroquine inhibites Rab11 mediated E-cadherin transport in collective colon cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1582.

Potent Imidazothiazole-based Inhibitor of BRAF V600E Overcomes Acquired Resistance via Inhibition of RAF Dimerization in PLX4032-resistant Melanoma.

The B-raf proto-oncogene, serine/threonine kinase (BRAF) V600E mutation is frequent in patients with advanced melanoma. PLX4032, an inhibitor of BRAFV600E kinase, is effective for the treatment of melanoma in BRAF V600E-positive patients; however, resistance eventually develops due to paradoxical activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinases (ERK) pathway resulting from RAF dimerization. In this study, we investigated the inhibitory effects of a novel imidazothiazole-based compound, KS28, on RAF dimerization and resistance to PLX4032 in melanoma. The effects of KS28 were examined by immunoblotting, cell viability, terminal deoxynucleotidyl transferase dUTP nick-end labeling, reporter-gene, and soft-agar assays. KS28 treatment inhibited RAF dimerization in PLX4032-resistant A375 (A375R) cells, leading to suppression of the MEK/ERK pathway. In addition, KS28 reduced activator protein 1 transactivation in A375R cells, reduced cell viability, and increased DNA fragmentation. Moreover, treatment with KS28 suppressed anchorage-independent growth of A375R cells. Similarly, in an orthotopic tumor xenograft model, KS28 treatment suppressed the growth of tumors formed by A375R cells in BALB/c mice. KS28 plays a vital role in overcoming PLX4032 resistance in melanoma by down-regulating the MEK/ERK pathway.

Proteomic analysis reveals dual requirement for Grb2 and PLCγ1 interactions for BCR-FGFR1-Driven 8p11 cell proliferation.

Translocation of Fibroblast Growth Factor Receptors (FGFRs) often leads to aberrant cell proliferation and cancer. The BCR-FGFR1 fusion protein, created by chromosomal translocation t(8;22)(p11;q11), contains Breakpoint Cluster Region (BCR) joined to Fibroblast Growth Factor Receptor 1 (FGFR1). BCR-FGFR1 represents a significant driver of 8p11 myeloproliferative syndrome, or stem cell leukemia/lymphoma, which progresses to acute myeloid leukemia or T-cell lymphoblastic leukemia/lymphoma. Mutations were introduced at Y177F, the binding site for adapter protein Grb2 within BCR; and at Y766F, the binding site for the membrane associated enzyme PLCγ1 within FGFR1. We examined anchorage-independent cell growth, overall cell proliferation using hematopoietic cells, and activation of downstream signaling pathways. BCR-FGFR1-induced changes in protein phosphorylation, binding partners, and signaling pathways were dissected using quantitative proteomics to interrogate the protein interactome, the phosphoproteome, and the interactome of BCR-FGFR1. The effects on BCR-FGFR1-stimulated cell proliferation were examined using the PLCγ1 inhibitor U73122, and the irreversible FGFR inhibitor futibatinib (TAS-120), both of which demonstrated efficacy. An absolute requirement is demonstrated for the dual binding partners Grb2 and PLCγ1 in BCR-FGFR1-driven cell proliferation, and new proteins such as ECSIT, USP15, GPR89, GAB1, and PTPN11 are identified as key effectors for hematopoietic transformation by BCR-FGFR1.

MET Inhibition Sensitizes Rhabdomyosarcoma Cells to NOTCH Signaling Suppression.

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma. The Fusion-Positive (FP) subtype expresses the chimeric protein PAX3-FOXO1 (P3F) while the Fusion-Negative (FN) is devoid of any gene translocation. FP-RMS and metastatic FN-RMS are often unresponsive to conventional therapy. Therefore, novel therapeutic approaches are needed to halt tumor progression. NOTCH signaling has oncogenic functions in RMS and its pharmacologic inhibition through γ-secretase inhibitors blocks tumor growth in vitro and in vivo. Here, we show that NOTCH signaling blockade resulted in the up-regulation and phosphorylation of the MET oncogene in both RH30 (FP-RMS) and RD (FN-RMS) cell lines. Pharmacologic inhibition of either NOTCH or MET signaling slowed proliferation and restrained cell survival compared to control cells partly by increasing Annexin V and CASP3/7 activation. Co-treatment with NOTCH and MET inhibitors significantly amplified these effects and enhanced PARP1 cleavage in both cell lines. Moreover, it severely hampered cell migration, colony formation, and anchorage-independent growth compared to single-agent treatments in both cell lines and significantly prevented the growth of FN-RMS cells grown as spheroids. Collectively, our results unveil the overexpression of the MET oncogene by NOTCH signaling targeting in RMS cells and show that MET pathway blockade sensitizes them to NOTCH inhibition.

Independent effects of Src kinase and podoplanin on anchorage independent cell growth and migration.

The Src tyrosine kinase is a strong tumor promotor. Over a century of research has elucidated fundamental mechanisms that drive its oncogenic potential. Src phosphorylates effector proteins to promote hallmarks of tumor progression. For example, Src associates with the Cas focal adhesion adaptor protein to promote anchorage independent cell growth. In addition, Src phosphorylates Cas to inducePdpnexpression to promote cell migration. Pdpn is a transmembrane receptor that can independently increase cell migration in the absence of oncogenic Src kinase activity. However, to our knowledge, effects of Src kinase activity on anchorage independent cell growth and migration have not been examined in the absence of Pdpn expression. Here, we analyzed the effects of an inducible Src kinase construct in knockout cells with and without exogenous Pdpn expression on cell morphology migration and anchorage independent growth. We report that Src promoted anchorage independent cell growth in the absence of Pdpn expression. In contrast, Src was not able to promote cell migration in the absence of Pdpn expression. In addition, continued Src kinase activity was required for cells to assume a transformed morphology since cells reverted to a nontransformed morphology upon cessation of Src kinase activity. We also used phosphoproteomic analysis to identify 28 proteins that are phosphorylated in Src transformed cells in a Pdpn dependent manner. Taken together, these data indicate that Src utilizes Pdpn to promote transformed cell growth and motility in complementary, but parallel, as opposed to serial, pathways.

Fetuin-A Promotes 3-Dimensional Growth in LNCaP Prostate Cancer Cells by Sequestering Extracellular Vesicles to Their Surfaces to Act as Signaling Platforms.

The present studies were conducted to evaluate key serum proteins and other components that mediate anchorage-independent growth (3-D growth) of LNCaP prostate cancer cells as spheroids. The cells were cultured on ultra-low attachment plates in the absence and presence of fetuin-A and with or without extracellular vesicles. The data show that fetuin-A (alpha 2HS glycoprotein) is the serum protein that mediates 3-D growth in these cells. It does so by sequestering extracellular vesicles of various sizes on the surfaces of rounded cells that grow as spheroids. These vesicles in turn transmit growth signals such as the activation of AKT and MAP kinases in a pattern that differs from the activation of these key growth signaling pathways in adherent and spread cells growing in 2-D. In the process of orchestrating the movement and disposition of extracellular vesicles on these cells, fetuin-A is readily internalized in adhered and spread cells but remains on the surfaces of non-adherent cells. Taken together, our studies suggest the presence of distinct signaling domains or scaffolding platforms on the surfaces of prostate tumor cells growing in 3-D compared to 2-D.

HspBP1 is a dual function regulatory protein that controls both DNA repair and apoptosis in breast cancer cells

The Hsp70-binding protein 1 (HspBP1) belongs to a family of co-chaperones that regulate Hsp70 activity and whose biological significance is not well understood. In the present study, we show that when HspBP1 is either knocked down or overexpressed in BRCA1-proficient breast cancer cells, there were profound changes in tumorigenesis, including anchorage-independent cell growth in vitro and in tumor formation in xenograft models. However, HspBP1 did not affect tumorigenic properties in BRCA1-deficient breast cancer cells. The mechanisms underlying HspBP1-induced tumor suppression were found to include interactions with BRCA1 and promotion of BRCA1-mediated homologous recombination DNA repair, suggesting that HspBP1 contributes to the suppression of breast cancer by regulating BRCA1 function and thereby maintaining genomic stability. Interestingly, independent of BRCA1 status, HspBP1 facilitates cell survival in response to ionizing radiation (IR) by interfering with the association of Hsp70 and apoptotic protease-activating factor-1. These findings suggest that decreased HspBP1 expression, a common occurrence in high-grade and metastatic breast cancers, leads to genomic instability and enables resistance to IR treatment.

The α2,8-sialyltransferase 6 (St8sia6) localizes in the ER and enhances the anchorage-independent cell growth in cancer

Sialylation, the final stage of post-translational modification of proteins, is achieved in the Golgi apparatus and is related to the malignant phenotype of cancer. Disialylation of ganglioside (GD3) by St8sia1 and polysialylation by St8sia2 and 4 have been shown to be related to malignant phenotypes; however, di/oligosialylation by St8sia6 is still unknown. In this study, we analyzed the malignant phenotype of St8sia6 and found that upregulation of St8sia6 in melanoma B16 cells increased anchorage-independent cell growth, which was not due to sialic acid cleavage by a sialidase. Moreover, unlike other sialyltransferases, St8sia6 localized to the endoplasmic reticulum (ER). We found that the localization to the Golgi apparatus could be regulated by swapping experiments using St8sia2; however, the malignant phenotype did not change. These data demonstrate that the enhancement of anchorage-independent cell growth by St8sia6 is not due to its localization of ER, but is due to the expression of the protein itself.

NRG1/ERBB3/ERBB2 Axis Triggers Anchorage-Independent Growth of Basal-like/Triple-Negative Breast Cancer Cells.

Simple SummaryBreast cancer is a heterogeneous disease, categorized into distinct subgroups with different clinical prognoses and treatment strategies. This study aimed to evaluate the role of ERBB3 in different molecular subtypes of breast cancers. Despite ERBB3/HER3 and its partner ERBB2/HER2 showing low expression levels in basal-like/triple-negative breast cancers, stratification of basal-like patients according to ERBB3 mRNA expression levels highlighted a correlation between higher ERBB3 levels and shorter relapse-free patients’ survival. In vitro analyses unveiled that the activation of the NRG1/ERBB3/ERBB2 axis robustly induces anchorage-independent growth of basal-like/triple-negative breast cancer cellular models, without significant effects on cell proliferation, differentiation, and migration in adhesion. Overall, our data suggest that ERBB2/ERBB3 plays an oncogenic role in basal-like/triple-negative breast cancer patients, suggesting its neutralization as a therapeutic strategy for these breast cancer subtypes, which today have very limited treatment opportunities.ERBB3, also known as HER3, is a tyrosine kinase transmembrane receptor of the ERBB family. Upon binding to neuregulin 1 (NRG1), ERBB3 preferentially dimerizes with HER2 (ERBB2), in turn inducing aggressive features in several cancer types. The analysis of a dataset of breast cancer patients unveiled that higher ERBB3 mRNA expression correlates with shorter relapse-free survival in basal-like breast cancers, despite low ERBB3 expression in this breast cancer subtype. Administration of neuregulin 1 beta (NRG1β) significantly affected neither cellular proliferation nor the basal migratory ability of basal-like/triple-negative quasi-normal MCF10A breast cells, cultured in mono-layer conditions. Furthermore, no significant regulation in cell morphology or in the expression of basal/myoepithelial and luminal markers was observed upon stimulation with NRG1β. In non-adherent conditions, NRG1β administration to MCF10A cells did not significantly influence cell survival; however, it robustly induced cell growth as spheroids (3D growth). Intriguingly, a remarkable upregulation of ERBB3 and ERBB2 protein abundance was observed in 3D compared to 2D cell cultures, and NRG1β-induced 3D cell growth was efficiently prevented by the anti-HER2 monoclonal antibody pertuzumab. Similar results were obtained by the analysis of basal-like/triple-negative breast cancer cellular models, MDA-MB-468 and MDA-MB-231 cells, in which NRG1β induced anchorage-independent cell growth that in turn was prevented or reduced by the simultaneous administration of anti-HER2 neutralizing antibodies. Finally, the ability of pertuzumab in suppressing NRG1β-induced 3D growth was also evaluated and confirmed in MCF10A engineered with HER2-overexpression. We suggest that the NRG1/ERBB3/ERBB2 pathway promotes the anchorage-independent growth of basal-like breast cancer cells. Importantly, we provide evidence that ERBB2 neutralization, in particular by pertuzumab, robustly inhibits this process. Our results pave the way towards the development of novel anticancer strategies for basal-like breast cancer patients based on the interception of the NRG1/ERBB3/ERBB2 signaling axis.

Hyperforin and Myrtucommulone Derivatives Act as Natural Modulators of Wnt/β-Catenin Signaling in HCT116 Colon Cancer Cells

The therapeutic activities of natural plant extracts have been well known for centuries. Many of them, in addition to antiviral and antibiotic effects, turned out to have anti-tumor activities by targeting different signaling pathways. The canonical Wnt pathway represents a major tumorigenic pathway deregulated in numerous tumor entities, including colon cancer. Here, we investigated the acylphloroglucinols hyperforin (HF) from St. John’s wort (Hypericum perforatum L.) and myrtucommulone A (MC A) from myrtle (Myrtus communis) and semi-synthetic derivatives thereof (HM 177, HM 297, HM298) for their effects on Wnt/β-catenin signaling. None of these substances revealed major cytotoxicity on STF293 embryonic kidney and HCT116 colon carcinoma cells at concentrations up to 10 μM. At this concentration, HF and HM 177 showed the strongest effect on cell proliferation, whereas MC A and HM 177 most prominently inhibited anchorage-independent growth of HCT116 cells. Western blot analyses of active β-catenin and β-catenin/TCF reporter gene assays in STF293 cells revealed inhibitory activities of HF, MC A and HM 177. In line with this, the expression of endogenous Wnt target genes, Axin and Sp5, in HCT116 cells was significantly reduced. Our data suggest that the acylphloroglucinols hyperforin, myrtucommulone A and its derivative HM 177 represent potential new therapeutic agents to inhibit Wnt/β-catenin signaling in colon cancer.

The Role of SOD2 in Migration and Anchorage-Independent Growth of SKOV3 Ovarian Cancer Cells

The 5-year survival rates for ovarian cancer are 93%, 75%, and 30% for localized, regional, and distant tumors, respectively. These vast differences in survival rates underscore the need to identify novel therapeutic targets that are effective at different stages of tumor progression to better treat all patients diagnosed with this deadly disease. Manganese superoxide dismutase (SOD2) is a mitochondrial antioxidant enzyme responsible for eliminating superoxide and preventing oxidative damage to the mitochondria. Recent studies have implicated changes in SOD2 expression levels in multiple cancers, including breast, colorectal, prostate, and head and neck. While studies have begun to unravel the role of SOD2 in ovarian cancer, no one has looked at the specific role SOD2 plays in distinct stages of tumor progression of ovarian cancer. Here, we report SOD2 deficiency (accomplished through utilizing shRNA techniques) results in increased invasive and migratory ability of SKOV3 cells, a commonly used ovarian adenocarcinoma cell line. In contrast, SOD2-deficient SKOV3 cells display abrogated anchorage-independent growth in soft agar. These studies in SKOV3 cells indicate that SOD2 expression hampers invasion and migration critical for early tumor initiation but helps maintain anchorage-independent growth necessary for ovarian cancer metastases. In aggregate, SOD2 plays a context-dependent role in ovarian cancer progression and its utility as a therapeutic target for later-stage anchorage-independent ovarian cancer cells should be further explored. (First online: March 1, 2022)

Abstract P4-02-14: Breast cancer cell/adipocyte crosstalk in obesity hampers the efficacy of tamoxifen

Abstract Despite the improvements in the outcomes of breast cancer patients following the implementation of endocrine therapy into daily oncology practice, the development of ‘de novo’ or acquired resistance to these treatments has become a major limitation. To date, several hallmarks of hormonal resistance have been proposed, including ERα loss or mutations, activation of growth factor-mediated signaling pathways, and alterations of key cell cycle checkpoints. More recently, it was proven that a reduced response to these therapeutic regimens may rely not only on tumor-cell intrinsic factors, but it is also dependent on the surrounding microenvironment and on the host characteristics. Indeed, clinical studies have suggested that obesity, in addition to promoting breast cancer development and progression, represents a heavyweight player driving breast cancer endocrine resistance. However, the influence of adipocytes and their secretome on the efficacy of hormone therapy has not properly investigated. In a model of co-culture, we demonstrated that the anti-proliferative effects of Tamoxifen on anchorage-dependent and anchorage-independent growth of MCF-7 breast cancer cells were counteracted by conditioned media (CM) released by fully differentiated 3T3-L1 cells (3T3-L1 A), a widely used 'in vitro' model of white adipocytes. Moreover, pre-incubation of MCF-7 cells with adipocyte-derived CM reduced their sensitivity to the inhibitory effects of Tamoxifen on motility, as evidenced by wound-healing and transmigration assays. Interestingly, the growth and the motility of Tamoxifen-resistant breast cancer cells were dramatically increased in the presence of adipocyte-derived CM, further suggesting a supportive role of adipocyte secretome in the development and progression of endocrine-resistant phenotypes. Quantitative transcriptome profiling of Tamoxifen-resistant cells revealed 917 differentially expressed genes (FDR ≤ 0.05), of which 243 were upregulated and 180 were downregulated in response to adipocyte-CM. Among these major modifications, the most significant down-regulated gene was Thioredoxin-interacting protein (TXNIP, 5-fold), a tumor suppressor participating in the metabolic reprogramming and oxidative stress in various solid cancers. These results well correlated with data obtained in retrospective analyses showing that low TXNIP expression in breast cancer patients was associated with a statistically significant poorer recurrence-free (RFS), overall (OS), post-progression (PPS) and distant metastasis-free (DMFS) survival compared to high TXNIP-expressing patients. This relevant discrimination is also achieved in tamoxifen-treated patients, highlighting a role of TXNIP for identifying patients at high risk of progression. These findings bring insights on adipocytes and mammary cancer cell interactions during Tamoxifen therapy, particularly in overweight/obese people and add evidence to advocating obesity as a parameter to be considered when identifying treatments for patients with ER-positive breast cancer. Citation Format: Amanda Caruso, Luca Gelsomino, Giusi La Camera, Cinzia Giordano, Salvatore Panza, Daniela Bonofiglio, Stefania Catalano, Sebastiano Andò, Ines Barone. Breast cancer cell/adipocyte crosstalk in obesity hampers the efficacy of tamoxifen [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-14.

Aberrant Expression and Subcellular Localization of ECT2 Drives Colorectal Cancer Progression and Growth.

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a Kras G12D Apc-null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. SIGNIFICANCE: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis.

Knockdown Wiskott-Aldrich syndrome protein family member 3 (WASF3) inhibits colorectal cancer metastasis and sensitizes to cisplatin through targeting ZNF471

Colorectal cancer (CRC) is a heterogeneous cancer, and many risk factors for colorectal cancer have been established. For CRC metastasis, tumor cell migration, adhesion as well as invasion are important processes. WiskottAldrich syndrome protein family member 3 (WASF3) is necessary for metastasis of various types of cancers. However, its role in CRC progression has not been fully elucidated. This study examined the in vitro functional roles of WASF3 in the CRC and explored the underlying molecular mechanisms. We used siRNA-WASF3 to gene silence WASF3 in colon cancer cell (HCT116) in vitro. The effects of WASF3 silencing on HCT116 cell apoptosis, proliferation, migration, as well as invasion were assessed by flow cytometry, CCK-8, and transwell assays. ZNF471 protein expressions were detected by immunofluorescence staining and RT-PCR. Moreover, the effects of ZNF471 were studied on a series of in vitro antitumor-promoting assays using HCT116. WASF3 knockdown expression using small interfering RNA (siRNA) ameliorated CRC cell proliferation, anchorage-independent growth, invasion, and metastasis. Furthermore, we observed that WASF3 contributed to upregulating the metastasis signaling pathway through inhibiting the expression of ZNF471. Our study suggests that targeting WASF3 signaling might be a novel therapeutic strategy for treating CRC.

Expression of CD98hc in Pancreatic Cancer and Its Role in Cancer Cell Behavior.

Background: Cluster of differentiation 98 heavy chain (CD98hc) is a transmembrane protein, which functions both as a coreceptor of ß-integrins, enhancing intracellular integrin-dependent downstream signaling, and as a transporter of branched-chain and aromatic amino acids. As such, it is pivotal in cell cycle regulation and protection of oxidative, nutritional and DNA replication stress. Overexpression of CD98hc occurs widely in cancer cells and is associated with poor clinical prognosis. The role of CD98hc in pancreatic cancer remains to be elucidated. The aim of this study was to determine the expression of CD98hc in pancreatic ductal adenocarcinoma and to define its potential functional role in cancer cell biology.Methods: Immunohistochemical staining for CD98hc was performed on 222 tissue samples of patients with pancreatic ductal adenocarcinoma. The pancreatic cancer cell lines PANC-1 and BxPC-3 were used to determine the effect of CD98hc expression on cancer cell behavior using cell adhesion, cell trans-migration and cell spreading assays. Flow cytometry was performed to study the rate of apoptosis after detachment or serum starvation. shRNA-lentiviral constructs were used to knock down or reconstitute full length or mutated CD98hc.Results: Up to 20% of pancreatic ductal adenocarcinomas express CD98hc in the acinar cells (13%) and islet cells (20%) embedded in tumor tissue. Although expression of CD98hc in tumor tissue was not associated with a particular tumor stage or grade, our data show a trend towards longer overall survival of pancreatic cancer patients without CD98hc expression as compared to those with immunohistochemical positivity. In vitro downregulation of CD98hc in the pancreatic cancer cell lines PANC-1 and BxPC-3 significantly inhibits cell proliferation (p<0.05), self-renewal (p<0.05) and anchorage-independent growth (p<0.05).Conclusion: CD98hc is expressed in a remarkable percentage of pancreatic ductal adenocarcinomas. Due to its important role in cell behavior and malignant cell transformation, it may be a promising molecular target for potential new therapeutic approaches in pancreatic cancer in the future.

CES2 sustains HNF4α expression to promote pancreatic adenocarcinoma progression through an epoxide hydrolase-dependent regulatory loop

ObjectiveIntra-tumoral expression of the serine hydrolase carboxylesterase 2 (CES2) contributes to the activation of the pro-drug irinotecan in pancreatic ductal adenocarcinoma (PDAC). Given other potential roles of CES2, we assessed its regulation, downstream effects, and contribution to tumor development in PDAC. MethodsAssociation between the mRNA expression of CES2 in pancreatic tumors and overall survival was assessed using The Cancer Genome Atlas. Cell viability, clonogenic, and anchorage-independent growth assays as well as an orthotopic mouse model of PDAC were used to evaluate the biological relevance of CES2 in pancreatic cancer. CES2-driven metabolic changes were determined by untargeted and targeted metabolomic analyses. ResultsElevated tumoral CES2 mRNA expression was a statistically significant predictor of poor overall survival in PDAC patients. Knockdown of CES2 in PDAC cells reduced cell viability, clonogenic capacity, and anchorage-independent growth in vitro and attenuated tumor growth in an orthotopic mouse model of PDAC. Mechanistically, CES2 was found to promote the catabolism of phospholipids resulting in HNF4α activation through a soluble epoxide hydrolase (sEH)-dependent pathway. Targeting of CES2 via siRNA or small molecule inhibitors attenuated HNF4α protein expression and reduced gene expression of classical/progenitor markers and increased basal-like markers. Targeting of the CES2-sEH-HNF4α axis using small molecule inhibitors of CES2 or sEH reduced cell viability. ConclusionsWe establish a novel regulatory loop between CES2 and HNF4α to sustain the progenitor subtype and promote PDAC progression and highlight the potential utility of CES2 or sEH inhibitors for the treatment of PDAC as part of non-irinotecan-containing regimens.

Catalase Overexpression Drives an Aggressive Phenotype in Glioblastoma.

Glioblastoma remains the deadliest form of brain cancer, largely because these tumors become resistant to standard of care treatment with radiation and chemotherapy. Intracellular production of reactive oxygen species (ROS) is necessary for chemo- and radiotherapy-induced cytotoxicity. Here, we assessed whether antioxidant catalase (CAT) affects glioma cell sensitivity to temozolomide and radiation. Using The Cancer Genome Atlas database, we found that CAT mRNA expression is upregulated in glioma tumor tissue compared with non-tumor tissue, and the level of expression negatively correlates with the overall survival of patients with high-grade glioma. In U251 glioma cells, CAT overexpression substantially decreased the basal level of hydrogen peroxide, enhanced anchorage-independent cell growth, and facilitated resistance to the chemotherapeutic drug temozolomide and ionizing radiation. Importantly, pharmacological inhibition of CAT activity reduced the proliferation of glioma cells isolated from patient biopsy samples. Moreover, U251 cells overexpressing CAT formed neurospheres in neurobasal medium, whereas control cells did not, suggesting that the radio- and chemoresistance conferred by CAT may be due in part to the enrichment of glioma stem cell populations. Finally, CAT overexpression significantly decreased survival in an orthotopic mouse model of glioma. These results demonstrate that CAT regulates chemo- and radioresistance in human glioma.