Aggressive Cancer Phenotype Research Articles

Abstract P5-05-07: An IGF-1R-mTORC1-SRPK2 signaling axis contributes to FASN regulation in breast cancer

Abstract BACKGROUND: FASN expression is associated with a more aggressive breast cancer phenotype and is both transcriptionally and post-transcriptionally regulated downstream of receptor tyrosine kinase signaling pathways. Moreover, 2nd and 3rd generation FASN inhibitors are showing promise for the treatment of breast cancer in clinical trials. Lipogenic transcripts, such as FASN, can be post-transcriptionally regulated through pre-mRNA splicing mediated through serine arginine rich protein kinases (SRPKs) and their respected substrates, serine-arginine rich splicing factors (SRSFs). Recent work has highlighted the mTORC1 signaling pathway as an upstream inducer of lipogenic pre-mRNA splicing, however, the role of extracellular environmental cues, such as growth factors and their respected receptors have not been explored. Here, an IGF-1-mTORC1-SRPK2 axis is demonstrated in the FASN regulation through SRSF-1 in breast cancer. METHODS: MDA-MB-231, MCF-7 breast cancer or MCF-10A non-transformed cells were exposed to IGF-1 followed by siRNA knockdown of IGF-1R, SRPK2, or SRSF-1. FASN expression was quantified by RT-qPCR or western blot analysis and de novo palmitate was measured by U-13C glucose incorporation followed by GS-MS. For mRNA stability, cells were pretreated with actinomycin-D with either vehicle or SRPK2 inhibitor for various timepoints followed by RT-qPCR for lipogenic and glycolytic mRNA abundance. eGFP-SRSF-1 was transfected in MDA-MB-231 and MCF-7 to visualize SRSF-1 localization in response to mTORC1 inhibition and/or SRPK2 knockdown and visualized by fluorescence microscopy. For intron retention, RT-PCR was performed with FASN intron and exon specific primers and resolved on a 2.5% agarose gel. RESULTS: Both IGF-1R and SRPK2 RNAi mediated knockdown significantly reduced FASN mRNA and protein and de novo synthesized palmitate levels. Similar results were obtained with mTORC1 inhibition. IGF-1 promoted the stabilization of FASN mRNA as well as reduced intron retention. This reduction of intron retention upon IGF-1 was abolished by SRPK2 knockdown. Additionally, IGF-1 contributed to a more diffuse localization in the nucleoplasm of SRSF-1, which become more retained in nuclear speckles upon both SRPK2 knockdown and mTORC1 inhibition. CONCLUSION: These current findings establish a potential IGF-1-mTORC1-SRPK2 axis in breast cancer that contributes to metabolic programming through FASN. More specifically, SRSF-1 is the potential mediator of FASN expression through this pathway, which could be a potential therapeutic target for breast cancers that overexpress FASN and components of the IGF-1R signaling axis. Citation Format: Bryan Mcclellan, Paul Gries, Brittany Harlow, Andrew J Brenner, Stefano Tiziani, Christopher Jolly, Linda deGraffenried. An IGF-1R-mTORC1-SRPK2 signaling axis contributes to FASN regulation in breast cancer [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 P5-05-07.

Abstract ES1-2: Genomics of HER2+ breast cancer

Abstract Human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in about 20% ofall breast cancers and, when untreated, is the most aggressive breast cancer phenotype. The prognosis of patients with HER2-positive breast cancer has been markedly improved thanks to the effectiveness of anti-HER2 therapies. However, we still overtreat a high proportion of patients with toxic chemotherapy +antiHER2 combinations, while others still develop recurrent disease despite these complex regimens. Both the intrinsic complexity of HER2-positive tumors and the properties of its tumor microenvironment likely contribute to differences in response toanti-HER2 targeted therapies. HER2-positive breast cancer is highly heterogeneous, and all the intrinsic molecular subtypes can be identified within HER2-positive tumors by gene expression. HER2-enriched subtype has been systematically associated with higher pCR rates to anti-HER2 targeted therapies across multiple trials in early stages. Genomically, the HER2-enriched subtype is the most HER2 oncogene addicted of the subtypes, which likely explains the high sensitivity to anti-HER2 therapies. However, this may also explain why a HER2-enriched tumor with residual disease after anti-HER2 treatments carries a particularly poor prognosis. Conversely, both tumor-infiltrating lymphocytes (TILs) and RNA-based immune-signatures are significantly and independently associated with a higher likelihood of pCR and better survival outcomes in patients with HER2-positive disease. Thus, integrating the prognostic information provided by clinical factors, genomic subtype, and immune-related signatures is essential to improve the design of future clinical trials and establish new and better treatment algorithms for HER2-positive breast cancer treatment. Citation Format: C Perou. Genomics of HER2+ breast cancer. [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 ES1-2.

The Role of Hypoxia in Endometrial Cancer.

Endometrial cancer represents the most frequent neoplasia from the corpus uteri and comprises the 14th leading cause of death in women worldwide. Risk factors that contribute to the disease include early menarche, late menopause, nulliparity, and menopausal hormone use, as well as hypertension and obesity comorbidities. The clinical effectiveness of chemotherapy is variable, suggesting that novel molecular targeted therapies against specific cellular processes associated with the maintenance of cancer cell survival and therapy resistance ameliorate the rates of success in endometrial cancer treatment. In the course of tumor growth, cancer cells must adapt to decreased oxygen availability in the microenvironment by upregulation of hypoxia-inducible factors, which orchestrate the activation of a transcriptional program leading to cell survival. During this adaptative process, the hypoxic cancer cells may acquire invasive and metastatic properties as well as increased cell proliferation and resistance to chemotherapy, enhanced angiogenesis, vasculogenic mimicry, and maintenance of cancer cell stemness, which contribute to more aggressive cancer phenotypes. Several studies have shown that hypoxia-inducible factor 1 alpha (HIF-1α) protein is aberrantly overexpressed in many solid tumors of the breast, prostate, ovarian, bladder, colon, brain, and pancreas. Thus, it has been considered an important therapeutic target. Here, we reviewed the current knowledge of the relevant roles of cellular hypoxia mechanisms and HIF-1α functions in diverse processes associated with endometrial cancer progression. In addition, we also summarize the role of microRNAs in the posttranscriptional regulation of protein-encoding genes involved in the hypoxia response in endometrial cancer. Finally, we pointed out the need for urgent targeted therapies to impair the cellular processes activated by hypoxia in the tumor microenvironment.

Neurturin promotes tumor cell motility and angiogenesis in colorectal cancer

Neurturin (NRTN) is one of the glial cell line-derived neurotrophic factor family ligands crucial for neuron growth, differentiation and maintenance. Recent studies showed NRTN promotes an aggressive pancreatic cancer phenotype, and predicts shorter survival in lung cancer patients. However, its expression and function in colorectal cancer (CRC) remain unclear. Herein, we found NRTN was enriched in CRC cells, and predicted poor patients outcomes. Upregulated NRTN enhanced the migration and invasion of CRC cells and vascularization of endothelial cells. In mechanism, NRTN promoted ZEB1/N-cadherin and vascular endothelial growth factor (VEGF)-A expression in CRC cells, which were responsible for tumor cell motility and angiogenesis, respectively. More importantly, NRTN inhibition prevented CRC metastasis and angiogenesis in vivo. In conclusion, NRTN promotes CRC cells motility and tumor angiogenesis via inducing ZEB1/N-cadherin and VEGF-A overexpression. It is a potential therapeutic target and negative prognostic biomarker for CRC patients.

SELENOF is a new tumor suppressor in breast cancer.

Epidemiological evidence has indicated an inverse association between selenium status and various types of cancer, including breast cancer. Selenoproteins are the primary mediators of selenium effects in human health. We have previously reported loss of heterozygosity in breast tumor samples of the gene for one of the selenoproteins, SELENOF. The function of SELENOF remains unclear and whether SELENOF levels impact breast cancer risk or outcome is unknown. The mining of breast cancer patient databases revealed that SELENOF mRNA is significantly lower in late-stage tumor samples and lower levels of SELENOF also predict poor patient outcome from breast cancer. Genetically manipulating SELENOF in human breast cancer cells or in the murine mammary gland by overexpression, silencing or knockout impacted cell viability by affecting both proliferation and cell death. Restoring SELENOF can attenuate a number of aggressive cancer phenotypes in breast cancer cells, including clonogenic survival, and enhance the response to drugs or radiation used in breast cancer therapy. Importantly, enhancing SELENOF expression reduced in vivo tumor growth in a murine xenograft model of breast cancer. These data indicate that SELENOF is a new tumor suppressor in breast cancer.

ABI1-based expression signature predicts breast cancer metastasis and survival.

Despite the current standard of care, breast cancer remains one of the leading causes of mortality in women worldwide, thus emphasizing the need for better predictive and therapeutic targets. ABI1 is associated with poor survival and an aggressive breast cancer phenotype, although its role in tumorigenesis, metastasis, and the disease outcome remains to be elucidated. Here, we define the ABI1‐based seven‐gene prognostic signature that predicts survival of metastatic breast cancer patients; ABI1 is an essential component of the signature. Genetic disruption of Abi1 in primary breast cancer tumors of PyMT mice led to significant reduction of the number and size of lung metastases in a gene dose‐dependent manner. The disruption of Abi1 resulted in deregulation of the WAVE complex at the mRNA and protein levels in mouse tumors. In conclusion, ABI1 is a prognostic metastatic biomarker in breast cancer. We demonstrate, for the first time, that lung metastasis is associated with an Abi1 gene dose and specific gene expression aberrations in primary breast cancer tumors. These results indicate that targeting ABI1 may provide a therapeutic advantage in breast cancer patients.

Chronoradiobiology of Breast Cancer: The Time Is Now to Link Circadian Rhythm and Radiation Biology.

Circadian disruption has been linked to cancer development, progression, and radiation response. Clinical evidence to date shows that circadian genetic variation and time of treatment affect radiation response and toxicity for women with breast cancer. At the molecular level, there is interplay between circadian clock regulators such as PER1, which mediates ATM and p53-mediated cell cycle gating and apoptosis. These molecular alterations may govern aggressive cancer phenotypes, outcomes, and radiation response. Exploiting the various circadian clock mechanisms may enhance the therapeutic index of radiation by decreasing toxicity, increasing disease control, and improving outcomes. We will review the body’s natural circadian rhythms and clock gene-regulation while exploring preclinical and clinical evidence that implicates chronobiological disruptions in the etiology of breast cancer. We will discuss radiobiological principles and the circadian regulation of DNA damage responses. Lastly, we will present potential rational therapeutic approaches that target circadian pathways to improve outcomes in breast cancer. Understanding the implications of optimal timing in cancer treatment and exploring ways to entrain circadian biology with light, diet, and chronobiological agents like melatonin may provide an avenue for enhancing the therapeutic index of radiotherapy.

MiR-182-5p Modulates Prostate Cancer Aggressive Phenotypes by Targeting EMT Associated Pathways.

Prostate cancer (PCa) is a clinically heterogeneous disease, where deregulation of epigenetic events, such as miRNA expression alterations, are determinants for its development and progression. MiR-182-5p, a member of the miR-183 family, when overexpressed has been associated with PCa tumor progression and decreased patients’ survival rates. In this study, we determined the regulatory role of miR-182-5p in modulating aggressive tumor phenotypes in androgen-refractory PCa cell lines (PC3 and DU-145). The transient transfection of the cell lines with miR-182-5p inhibitor and mimic systems, significantly affected cell proliferation, adhesion, migration, and the viability of the cells to the chemotherapeutic agents, docetaxel, and abiraterone. It also affected the protein expression levels of the tumor progression marker pAKT. These changes, however, were differentially observed in the cell lines studied. A comprehensive biological and functional enrichment analysis and miRNA/mRNA interaction revealed its strong involvement in the epithelial-mesenchymal transition (EMT) process; expression analysis of EMT markers in the PCa transfected cells directly or indirectly modulated the analyzed tumor phenotypes. In conclusion, miR-182-5p differentially impacts tumorigenesis in androgen-refractory PCa cells, in a compatible oncomiR mode of action by targeting EMT-associated pathways.

Extracellular Vesicles Derived-LAT1 mRNA as a Powerful Inducer of Colorectal Cancer Aggressive Phenotype.

Simple SummaryThe metastatic spread of tumor cells to the liver is one of the most common causes of mortality in CRC. Extracellular vesicles are currently considered vehicles of metastization, playing a role in the modification of the recipient cell’s phenotype. LAT1 and ASCT2 are amino acids transporters associated with increased proliferation in CRC. This study analyzed the effect of LAT1 and ASCT2 mRNAs derived from CRC-EVs in the phenotype modulation of recipient cells. In this study we demonstrate LAT1-EVs mRNA involvement in recipient cells’ phenotype modulation, conferring advantages in cell migration and proliferation.Colorectal cancer (CRC) is the third most common cancer in the world and represents the third most deadly tumor worldwide. About 15–25% of patients present metastasis in the moment of diagnosis, the liver being the most common site of metastization. Therefore, the development of new therapeutic agents is needed, to improve the patients’ prognosis. Amino acids transporters, LAT1 and ASCT2, are described as upregulated in CRC, being associated with a poor prognosis. Extracellular vesicles have emerged as key players in cell-to-cell communication due to their ability to transfer biomolecules between cells, with a phenotypic impact on the recipient cells. Thus, this study analyzes the presence of LAT1 and ASCT2 mRNAs in CRC-EVs and evaluates their role in phenotype modulation in a panel of four recipient cell lines (HCA-7, HEPG-2, SK-HEP-1, HKC-8). We found that HCT 116-EVs carry LAT1, ASCT2 and other oncogenic mRNAs being taken up by recipient cells. Moreover, the HCT 116-EVs’ internalization was associated with the increase of LAT1 mRNA in SK-HEP-1 cells. We also observed that HCT 116-EVs induce a higher cell migration capacity and proliferation of SK-HEP-1 and HKC-8 cells. The present study supports the LAT1-EVs’ mRNA involvement in cell phenotype modulation, conferring advantages in cell migration and proliferation.

Transglutaminase 2 Maintains Hepatocyte Growth Factor Signaling to Enhance the Cancer Cell Phenotype.

Transglutaminase 2 (TG2) is a key epidermal squamous cell carcinoma cancer cell survival protein. However, how TG2 maintains the aggressive cancer phenotype is not well understood. The present studies show that TG2, which is highly expressed in epidermal cancer stem-like cells (ECS cells), maintains hepatocyte growth factor (HGF) signaling to drive an aggressive ECS cell cancer phenotype. Inhibiting TG2 reduces MET tyrosine kinase receptor expression and activity and attenuates the cancer cell phenotype. Moreover, inhibition of TG2 or HGF/MET function reduces downstream MEK1/2 and ERK1/2 activity, and this is associated with reduced cancer cell spheroid formation, invasion, and migration, and reduced stem and EMT marker expression. Treatment of TG2 knockdown cells with HGF partially restores the aggressive cancer phenotype, confirming that MET signaling is downstream of TG2. MET knockout reduces ERK1/2 signaling, doubles the time to initial tumor appearance, and reduces overall tumor growth. These findings suggest that TG2 maintains HGF/MET and MAPK (MEK1/2 and ERK1/2) signaling to drive the aggressive ECS cell cancer phenotype and tumor formation, and that TG2-dependent MET signaling may be a useful anti-cancer target. IMPLICATIONS: TG2 is an important epidermal squamous cell carcinoma stem cell survival protein. We show that TG2 activates an HGF/MET, MEK1/2 ERK1/2 signaling cascade that maintains the aggressive cancer phenotype.

Abstract P096: Using CRISPR-Cas9 screens to identify microRNA involved in aggressive prostate cancer phenotypes

Abstract Background: Prostate cancer (PCa) is the most common cancer diagnosis in Canadian men. For many, the tumour can remain “dormant” negating a need for treatment, but if the tumour progresses and becomes metastatic, prognosis is poor. Despite the introduction of new treatments, metastatic PCa remains a lethal and incurable disease. This is invariably due to the onset of castration-resistance in advanced cases producing a more aggressive tumour with increased metastatic potential. We hypothesize that microRNA can promote these aggressive PCa phenotypes due to their ability to regulate diverse gene networks simultaneously. Methods: We have generated miRKOv2, the second version of our microRNA CRISPR Knockout library using the latest on- and off-target scoring algorithms and other microRNA-specific features for CRISPR-Cas9 guide RNA (gRNA) design. miRKOv2 will be used in three separate screens to identify microRNA that are essential for PCa growth, microRNA that are involved in PCa metastasis, and microRNA that can confer castration-resistance, respectively. Results: Next generation sequencing of the plasmid miRKOv2 library showed a median gRNA coverage of ~1100X. In silico comparisons to other microRNA libraries demonstrate that miRKOv2 is a superior sgRNA library. DU145 cells stably expressing Cas9 are highly active and proof of principle growth assays demonstrate that essential gene knockout and subsequent decrease in cell viability is detectable. Generation of a Cas9+ PCa cell line panel and screens are ongoing. Conclusion: miRKOv2 is an improved microRNA-focused CRISPR library with high gRNA coverage. Proof of principle assays demonstrate that knockout of essential genes results in a reduction in cell viability, indicating that a CRISPR dropout screen is appropriate for our in vitro model. This project aims to identify microRNA dependencies in metastatic and castration-resistant PCa that can be leveraged into new biomarkers and therapies. Citation Format: Jonathan Tak-Sum Chow, Daniel K. C. Lee, Martino Marco Gabra, Norman Fu, Keyue Chen, Leonardo Salmena. Using CRISPR-Cas9 screens to identify microRNA involved in aggressive prostate cancer phenotypes [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P096.

Abstract CC02-01: Regulation of FASN expression through a novel IGF-1R-mTORC1-SRPK2-SRSF1 pathway

Abstract BACKGROUND FASN expression is associated with a more aggressive breast cancer phenotype and is both transcriptionally and post-transcriptionally regulated downstream of receptor tyrosine kinase signaling pathways. Lipogenic transcripts, such as FASN, can be post-transcriptionally regulated through pre-mRNA splicing mediated through serine arginine rich protein kinases (SRPKs) and their respected substrates, serine-arginine rich splicing factors (SRSFs). Recent work has highlighted the mTORC1 signaling pathway as an upstream inducer of lipogenic pre-mRNA splicing, however, the role of extracellular environmental cues, such as growth factors and their respected receptors have not been explored. Here, an IGF-1-mTORC1-SRPK2 axis is demonstrated in the FASN regulation through SRSF-1 in breast cancer. METHODS: MDA-MB-231, MCF-7 breast cancer or MCF-10A non-transformed cells were exposed to IGF-1 followed by siRNA knockdown of IGF-1R, SRPK2, or SRSF-1. FASN expression was quantified by RT-qPCR or western blot analysis and de novo palmitate was measured by U-13C glucose incorporation followed by GS-MS. For mRNA stability, cells were pretreated with actinomycin-D with either vehicle or SRPK2 inhibitor for various timepoints followed by RT-qPCR for lipogenic and glycolytic mRNA abundance. eGFP-SRSF-1 was transfected in MDA-MB-231 and MCF-7 to visualize SRSF-1 localization in response to mTORC1 inhibition and/or SRPK2 knockdown and visualized by fluorescence microscopy. For intron retention, RT-PCR was performed with FASN intron and exon specific primers and resolved on a 2.5% agarose gel. RESULTS: Both IGF-1R and SRPK2 RNAi mediated knockdown significantly reduced FASN mRNA and protein and de novo synthesized palmitate levels. Similar results were obtained with mTORC1 inhibition. IGF-1 promoted the stabilization of FASN mRNA as well as reduced intron retention. This reduction of intron retention upon IGF-1 was abolished by SRPK2 knockdown. Additionally, IGF-1 contributed to a more diffuse localization in the nucleoplasm of SRSF-1, which become more retained in nuclear speckles upon both SRPK2 knockdown and mTORC1 inhibition. CONCLUSION These current findings establish a potential IGF-1-mTORC1-SRPK2 axis in breast cancer that contributes to metabolic programming through FASN. More specifically, SRSF-1 is the potential mediator of FASN expression through this pathway, which could be a potential therapeutic target for breast cancers that overexpress FASN and components of the IGF-1R signaling axis. Citation Format: Bryan Mcclellan, Paul Gries, Brittany Harlow, Andrew Brenner, Stefano Tiziani, Christopher Jolly, Linda deGraffenried. Regulation of FASN expression through a novel IGF-1R-mTORC1-SRPK2-SRSF1 pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr CC02-01.

The expression and significance of 8-hydroxydeoxyguanosine in breast cancer patients’ blood, urine and cancer tissue

Objective: To explore the expression and clinic significance of 8-OHdG in breast cancer. Methods: Pre-operative serum 8-OHdG levels were detected with an enzyme-linked immunosorbent assay in a well-defined series of 173 breast cancer patients. 8-OHdG expression in cancer cells from 150 of these patients was examined by immunohistochemistry. The HPLC-ECD method is used to determine 8-OHdG concentration in urine. Results: The serum 8-OHdG levels and immunohistochemical 8-OHdG expression were in concordance with each other (P < 0.05, r = 0.163). Breast cancer patients with negative 8-OHdG immunostaining show lower survival rate according to the multivariate analysis (P < 0.01). This observation was even more remarkable in ductal carcinomas (n = 140) patients (P < 0.001). A low serum 8-OHdG level was associated statistically significantly with lymphatic vessel invasion and a positive lymph node status. Comparison of 8-OHdG concentration in urine of breast cancer patients and healthy women was statistical significance (P < 0.01). Conclusion: Low serum 8-OHdG levels and a low immunohistochemical 8-OHdG expression were associated with an aggressive breast cancer phenotype. In addition, negative 8-OHdG immunostaining was an independent prognostic factor for breast cancer-specific death in breast carcinoma patients. Using 8-OHdG concentration in urine to predict DNA damage resulting from breast cancer can provide good biological indicators for detecting harm in early breast cancer.

Expression profile of tumour suppressor protein p53 and its regulator MDM2 in a cohort of breast cancer patients in a Tertiary Hospital in Ghana.

BackgroundInactivation or mutation of the tumour suppressor gene p53 or its regulator mouse double minute 2 (MDM2) is the commonest event in breast cancer. These altered genes usually express abnormally high levels of their proteins in many carcinomas. The phenotypic expression of p53 and MDM2 in breast cancer cases in our setting is not known. This study investigated the expression of the tumour suppressor protein p53 and its regulator MDM2, using immunohistochemistry in a Ghana breast cancer cohort.MethodA 9-year retrospective cross-sectional study on archived tissue blocks–formalin fixed paraffin embedded tissue (FFPE) was carried out. Demographic data were abstracted. Based on complete clinical data and availability of FFPE archived blocks 203 cases were selected for tissue micro array (TMA) construction. The TMA sections were subjected to immunohistochemistry (IHC) (ER, PR, HER2, p53, and MDM2). Expression of p53 and MDM2 were related to grade and molecular subtypes.ResultsThe age ranged from 17 to 92 years (mean = 49.34 ± 13.74). Most of the cases were high grade; grade II (34.9%) and grade III (55.7%). Fifty-four percent of the cases were triple negative. Invasive ductal carcinoma no special type was the commonest histotype (87.1%). Thirty-six percent (36%) of the cases expressed p53. Significant associations were found between p53 overexpression and histological grade (p = 0.034), triple negative (p = 0.0333) and luminal B (p<0.01) tumors. Most cases (93.1%) were negative for MDM2 expression. Significant association was found between MDM2 and HER2 over-expression as well as Ki-67. There was no significant positive correlation between MDM2 and p53 co-expression (p>0.05).ConclusionThe elevated level of p53 expression in the aggressive breast cancer phenotypes (high histological grade and triple negative) in our cohort suggest that P53 elevation may be a poor prognostic marker in our setting. High expression of MDM2 in our cohort with high Ki67; also in cases with Her2/neu overexpression known with predictable poor prognosis in the absence of target therapy suggest MDM2 may be associated with aggressive biological behaviour in our breast cancer cases. The non-significant association of p53 and MDM2 expression in the same cases as also documented by previous studies suggest independent genetic pathway in tumourigenesis.

Haploinsufficient tumor suppressor PRP4K is negatively regulated during epithelial-to-mesenchymal transition.

The pre‐mRNA processing factor 4 kinase (PRP4K, also known as PRPF4B) is an essential gene. However, reduced PRP4K expression is associated with aggressive breast and ovarian cancer phenotypes including taxane therapy resistance, increased cell migration and invasion in vitro, and cancer metastasis in mice. These results are consistent with PRP4K being a haploinsufficient tumor suppressor. Increased cell migration and invasion is associated with epithelial‐to‐mesenchymal transition (EMT), but how reduced PRP4K levels affect normal epithelial cell migration or EMT has not been studied. Depletion of PRP4K by small hairpin RNA (shRNA) in non‐transformed mammary epithelial cell lines (MCF10A, HMLE) reduced or had no effect on 2D migration in the scratch assay but resulted in greater invasive potential in 3D transwell assays. Depletion of PRP4K in mesenchymal triple‐negative breast cancer cells (MDA‐MB‐231) resulted in both enhanced 2D migration and 3D invasion, with 3D invasion correlated with higher fibronectin levels in both MDA‐MB‐231 and MCF10A cells and without changes in E‐cadherin. Induction of EMT in MCF10A cells, by treatment with WNT‐5a and TGF‐β1, or depletion of eukaryotic translation initiation factor 3e (eIF3e) by shRNA, resulted in significantly reduced PRP4K expression. Mechanistically, induction of EMT by WNT‐5a/TGF‐β1 reduced PRP4K transcript levels, whereas eIF3e depletion led to reduced PRP4K translation. Finally, reduced PRP4K levels after eIF3e depletion correlated with increased YAP activity and nuclear localization, both of which are reversed by overexpression of exogenous PRP4K. Thus, PRP4K is a haploinsufficient tumor suppressor negatively regulated by EMT, that when depleted in normal mammary cells can increase cell invasion without inducing full EMT.

Sulforaphane covalently interacts with the transglutaminase 2 cancer maintenance protein to alter its structure and suppress its activity.

Type 2 transglutaminase (TG2) functions as an important cancer cell survival protein in a range of cancers including epidermal squamous cell carcinoma. TG2 exists in open and closed conformations each of which has a distinct and mutually exclusive activity. The closed conformation has GTP-binding/GTPase activity while the open conformation functions as a transamidase to catalyze protein-protein crosslinking. GTP-binding/GTPase activity is required for TG2 maintenance of the aggressive cancer phenotype. Thus, identifying agents that convert TG2 from the closed to the open GTP-binding/GTPase inactive conformation is an important cancer prevention/treatment strategy. Sulforaphane (SFN) is an important diet-derived cancer prevention agent that is known to possess a reactive isothiocyanate group and has potent anticancer activity. Using a biotin-tagged SFN analog (Biotin-ITC) and kinetic analysis we show that SFN covalently and irreversibly binds to recombinant TG2 to inhibit transamidase activity and shift TG2 to an open/extended conformation, leading to a partial inhibition of GTP binding. We also show that incubation of cancer cells or cancer cell extract with Biotin-ITC results in formation of a TG2/Biotin-ITC complex and that SFN treatment of cancer cells inhibits TG2 transamidase activity and shifts TG2 to an open/extended conformation. These findings identify TG2 as a direct SFN anticancer target in epidermal squamous cell carcinoma.

Corrigendum to “Epigenetic silencing of miR-490-3p promotes development of an aggressive colorectal cancer phenotype through activation of the Wnt/β-catenin signaling pathway”. Cancer Letters, 376 (2016) 178-87

Corrigendum to “Epigenetic silencing of miR-490-3p promotes development of an aggressive colorectal cancer phenotype through activation of the Wnt/β-catenin signaling pathway”. Cancer Letters, 376 (2016) 178-87

Intact SMAD-4 is a predictor of increased locoregional recurrence in upfront resected pancreas cancer receiving adjuvant therapy.

Previous reports suggest that intact SMAD4 expression is associated with a locally aggressive pancreas cancer phenotype. The objectives of this work were to determine the frequency of intact SMAD4 and its association with patterns of recurrence in patients with upfront resected pancreas cancer receiving adjuvant therapy. A tissue microarray was constructed using resected specimens from patients who underwent upfront surgery and adjuvant gemcitabine with no neoadjuvant treatment for pancreas cancer. SMAD4 expression was determined by immunohistochemical staining. Associations of SMAD4 expression and clinicopathologic parameters with clinical outcomes were evaluated using Cox proportional hazard models. One hundred twenty-seven patients were included with a median follow up of 5.7 years. Most patients had stage ≥ pT3 tumors (75%) and pN1 (68%). All patients received adjuvant gemcitabine, and 79% of patients received adjuvant chemoradiotherapy. Ten (8%) patients had intact SMAD4 expression. Grade was the only clinicopathologic parameter statistically associated with SMAD4 expression (P=0.05). Median overall survival was 2.1 years. On univariate analysis, SMAD4 expression was associated with increased locoregional recurrence (hazard ratio 7.0, P<0.01, 95% confidence interval: 2.8-18.0) but not distant recurrence (P=0.06) or overall survival (P=0.73). On multivariable analysis, SMAD4 expression (hazard ratio 9.6, P<0.01, 95% confidence interval: 3.7-24.8) and adjuvant chemoradiotherapy (hazard ratio 0.3, P=0.01, 95% confidence interval: 0.1-0.8) were associated with higher and lower locoregional recurrence, respectively. In patients with upfront resected pancreas cancer, SMAD4 expression was associated with an increased risk of locoregional recurrence. Prospective evaluation of the frequency of SMAD4 expression and validation of its predictive utility is warranted.

Mucin-producing hamster cholangiocarcinoma cell line, Ham-2, possesses the aggressive cancer phenotypes with liver and lung metastases.

Cholangiocarcinoma (CCA) is an aggressive bile duct cancer. Opisthorchis viverrini (O. viverrini) infection is a significant cause of CCA in the Greater Mekong subregion. Currently, there is no standard chemotherapeutic regimen for CCA. A unique hamster carcinogenesis model of O. viverrini-associated CCA was established. Molecular targets identified from the hamster CCA-comparative model are valuable for target identification and validation. Hamster CCA was induced by the administration of O. viverrini metacercariae and N-nitrosodimethylamine. Hamster-derived cancer cells were isolated and continuously cultured for more than 6 months. Ham-2 cell line was established and characterized in vitro and in vivo. Ham-2 exhibited chromosome hyperploidy. A comparative study with previously established cell line, Ham-1, demonstrated that Ham-2 acquired slower growth, higher adhesion, higher migration, and resistance to doxorubicin and 5-fluorouracil (5-FU). In BALB/c Rag-2/Jak3 double-deficient (BRJ) mice, Ham-2 subcutaneous transplantation formed mucin-producing cancers, which morphologically resemble human tubular cholangiocarcinoma. Intravenous-injected Ham-2 established the metastatic nodules in the lungs and livers of BRJ mice. Altogether, a new hamster cholangiocarcinoma cell line, Ham-2, which acquired more aggressive phenotypes in vitro and in vivo, was established. This cell line might be a valuable tool for comparative drug target identification and validation.

A Claudin-Based Molecular Signature Identifies High-Risk, Chemoresistant Colorectal Cancer Patients.

Identifying molecular characteristics that are associated with aggressive cancer phenotypes through gene expression profiling can help predict treatment responses and clinical outcomes. Claudins are deregulated in colorectal cancer (CRC). In CRC, increased claudin-1 expression results in epithelial-to-mesenchymal transition and metastasis, while claudin-7 functions as a tumor suppressor. In this study, we have developed a molecular signature based on claudin-1 and claudin-7 associated with poor patient survival and chemoresistance. This signature was validated using an integrated approach including publicly available datasets and CRC samples from patients who either responded or did not respond to standard-of-care treatment, CRC cell lines, and patient-derived rectal and colon tumoroids. Transcriptomic analysis from a patient dataset initially yielded 23 genes that were differentially expressed along with higher claudin-1 and decreased claudin-7. From this analysis, we selected a claudins-associated molecular signature including PIK3CA, SLC6A6, TMEM43, and ASAP-1 based on their importance in CRC. The upregulation of these genes and their protein products was validated using multiple CRC patient datasets, in vitro chemoresistant cell lines, and patient-derived tumoroid models. Additionally, blocking these genes improved 5-FU sensitivity in chemoresistant CRC cells. Our findings propose a new claudin-based molecular signature that associates with poor prognosis as well as characteristics of treatment-resistant CRC including chemoresistance, metastasis, and relapse.