mCRC Cells Research Articles

Mechanism of bufalin inhibition of colon cancer liver metastasis by regulating M2-type polarization of Kupffer cells induced by highly metastatic colon cancer cells.

Patients with metastatic colorectal cancer often have poor outcomes, primarily due to hepatic metastasis. Colorectal cancer (CRC) cells have the ability to secrete cytokines and other molecules that can remodel the tumor microenvironment, facilitating the spread of cancer to the liver. Kupffer cells (KCs), which are macrophages in the liver, can be polarized to M2 type, thereby promoting the expression of adhesion molecules that aid in tumor metastasis. Our research has shown that huachanshu (with bufalin as the main active monomer) can effectively inhibit CRC metastasis. However, the underlying mechanism still needs to be thoroughly investigated. We have observed that highly metastatic CRC cells have a greater ability to induce M2-type polarization of Kupffer cells, leading to enhanced metastasis. Interestingly, we have found that inhibiting the expression of IL-6, which is highly expressed in the serum, can reverse this phenomenon. Notably, bufalin has been shown to attenuate the M2-type polarization of Kupffer cells induced by highly metastatic Colorectal cancer (mCRC) cells and down-regulate IL-6 expression, ultimately inhibiting tumor metastasis. In this project, our aim is to study how high mCRC cells induce M2-type polarization and how bufalin, via the SRC-3/IL-6 pathway, can inhibit CRC metastasis. This research will provide a theoretical foundation for understanding the anti-CRC effect of bufalin.

Transient receptor potential ankyrin 1 (TRPA1) mediates reactive oxygen species-induced Ca2+ entry, mitochondrial dysfunction, and caspase-3/7 activation in primary cultures of metastatic colorectal carcinoma cells

Colorectal carcinoma (CRC) represents the fourth most common cancer worldwide and is the third most common cause of malignancy-associated mortality. Distant metastases to the liver and lungs are the main drivers of CRC-dependent death. Pro-oxidant therapies, which halt disease progression by exacerbating oxidative stress, represent an antitumour strategy that is currently exploited by chemotherapy and ionizing radiation. A more selective strategy to therapeutically exploit reactive oxygen species (ROS) signaling would consist in targeting a redox sensor that is up-regulated in metastatic cells and is tightly coupled to the stimulation of cancer cell death programs. The non-selective cation channel, Transient Receptor Potential Ankyrin 1 (TRPA1), serves as a sensor of the cellular redox state, being activated to promote extracellular Ca2+ entry by an increase in oxidative stress. Recent work demonstrated that TRPA1 channel protein is up-regulated in several cancer types and that TRPA1-mediated Ca2+ signals can either engage an antiapoptotic pro-survival signaling pathway or to promote mitochondrial Ca2+ dysfunction and apoptosis. Herein, we sought to assess for the first time the outcome of TRPA1 activation by ROS on primary cultures of metastatic colorectal carcinoma (mCRC cells). We found that TRPA1 channel protein is up-regulated and mediates enhanced hydrogen peroxide (H2O2)-induced Ca2+ entry in mCRC cells as compared to non-neoplastic control cells. The lipid peroxidation product 4-hydroxynonenal (4-HNE) is the main ROS responsible for TRPA1 activation upon mCRC cell exposure to oxidative stress. TRPA1-mediated Ca2+ entry in response to H2O2 and 4-HNE results in mitochondrial Ca2+ overload, followed by mitochondrial depolarization and caspase-3/7 activation. Therefore, targeting TRPA1 could represent an alternative strategy to eradicate metastatic CRC by enhancing its sensitivity to oxidative stress.

320 (PB100) - A Novel Hybrid Bifunctional Alkylating Agent That Potently Suppresses the Growth of mCRC Cells Xenografts and Patient-Derived Organoids

Background: Hitherto metastatic colorectal cancer (mCRC) is the basis for the second-leading death of all cancers. As the first-line chemotherapy in mCRC treatment, an antiangiogenic combination of bevacizumab with FOLFOX4 improved the competence and overall survival (OS). We developed a new hybrid small molecule with dual properties as first-line therapy to study its synergistic effect on CRC cell lines and patient-derived organoids.

Graphene Oxide and Fluorescent-Aptamer-Based Novel Aptasensors for Detection of Metastatic Colorectal Cancer Cells.

Early diagnosis of metastatic colorectal cancer (mCRC) is extremely critical to improve treatment and extend survival. W3 is an aptamer that can specifically bind to mCRC cells with high affinity. Graphene oxide (GO) is a two-dimensional graphitic carbon nanomaterial, which has widely used in constructing biosensors. In this study, we have developed a no-wash fluorescent aptasensor for one-step and sensitive detection of mCRC LoVo cells. It is based on fluorescence resonance energy transfer (FRET) between GO and the W3 aptamer labeled with 5-carboxyfluorescein (FAM). GO can quench the green fluorescence of the FAM-labeled W3 (FAM-W3). In the presence of the target cells, FAM-W3 preferentially binds the target cells and detaches from the surface of GO, leading to the fluorescence of FAM recovery. It was demonstrated that the fluorescence recovery increases linearly in a wide range of 0~107 cells/mL (R2 = 0.99). The GO-based FAM-labeled W3 aptasensor (denoted as FAM-W3-GO) not only specifically recognizes mCRC cell lines (LoVo and HCT116), but also sensitively differentiates the target cells from mixed cells, even in the presence of only 5% of the target cells. Furthermore, FAM-W3-GO was applied to detect LoVo cells in human whole blood, which showed good reproducibility with an RSD range of 1.49% to 1.80%. Therefore, FAM-W3-GO may have great potential for early diagnosis of mCRC. This strategy of GO-based fluorescent aptasensor provides a simple, one-step, and highly sensitive approach for the detection of mCRC cells.

Store-Operated Ca2+ Entry Is Up-Regulated in Tumour-Infiltrating Lymphocytes from Metastatic Colorectal Cancer Patients.

Simple SummaryStore-operated Ca2+ entry (SOCE) has long been known to regulate the differentiation and effector functions of T cells as well as to be instrumental to the ability of cytotoxic T lymphocytes to target cancer cells. Currently, no information is available regarding the expression and function of SOCE in tumour-infiltrating lymphocytes (TILs) that have been expanded in vitro for adoptive cell therapy (ACT). This study provides the first evidence that SOCE is up-regulated in ex vivo-expanded TILs from metastatic colorectal cancer (mCRC) patients. The up-regulation of SOCE mainly depends on diacylglycerol kinase (DGK), which prevents the protein kinase C-dependent inhibition of Ca2+ entry in normal T cells. Of note, the pharmacological blockade of SOCE with the selective inhibitor, BTP-2, during target cell killing significantly increases cytotoxic activity at low TIL density, i.e., when TILs-mediated cancer cell death is rarer. This study, albeit preliminary, could lay the foundation to propose an alternative strategy to effect ACT. It has been shown that ex vivo-expanded TILs did not improve the disease-free survival rate in mCRC patients. Our results strongly suggest that pre-treating autologous TILs with a SOCE or DGK inhibitor before being infused into the patient could improve their cytotoxic activity against cancer cells.(1) Background: Store-operated Ca2+ entry (SOCE) drives the cytotoxic activity of cytotoxic T lymphocytes (CTLs) against cancer cells. However, SOCE can be enhanced in cancer cells due to an increase in the expression and/or function of its underlying molecular components, i.e., STIM1 and Orai1. Herein, we evaluated the SOCE expression and function in tumour-infiltrating lymphocytes (TILs) from metastatic colorectal cancer (mCRC) patients. (2) Methods: Functional studies were conducted in TILs expanded ex vivo from CRC liver metastases. Peripheral blood T cells from healthy donors (hPBTs) and mCRC patients (cPBTs) were used as controls. (3) Results: SOCE amplitude is enhanced in TILs compared to hPBTs and cPBTs, but the STIM1 protein is only up-regulated in TILs. Pharmacological manipulation showed that the increase in SOCE mainly depends on tonic modulation by diacylglycerol kinase, which prevents the protein kinase C-dependent inhibition of SOCE activity. The larger SOCE caused a stronger Ca2+ response to T-cell receptor stimulation by autologous mCRC cells. Reducing Ca2+ influx with BTP-2 during target cell killing significantly increases cytotoxic activity at low target:effector ratios. (4) Conclusions: SOCE is enhanced in ex vivo-expanded TILs deriving from mCRC patients but decreasing Ca2+ influx with BTP-2 increases cytotoxic activity at a low TIL density.

Abstract 3697: Exploiting patient-derived xenografts and organoids to tackle apoptotic dependencies in EGFR-inhibited colorectal tumours

Abstract Treatment with EGFR-targeted therapies, such as the EGFR monoclonal antibody cetuximab, has improved the outcome of patients with metastatic colorectal cancer (mCRC). However, EGFR inhibition is mainly cytostatic and does not lead to tumor eradication: subsets of drug-tolerant cancer cells often persist even after maximal response to therapy and foster residual disease, leading to relapse. Importantly, drug-tolerant cells are less prone to undergo apoptosis, and thus constitute a reservoir of treatment-refractory persisters that are difficult to extinguish. Here we aimed to investigate the mechanisms by which apoptosis escape mediates survival of cetuximab-tolerant mCRC cells as a means to find new therapeutic strategies for limiting residual disease and delaying disease recurrence. By performing an innovative functional assay named Dynamic BH3 Profiling in cetuximab-sensitive patient-derived organoids (PDOs), we found that antibody treatment increased the “apoptotic priming”, i.e. decreased the distance of cells from the apoptotic threshold without eliciting overt cell death. To start investigating the mechanisms of this increased apoptotic priming, protein levels of the major anti-apoptotic molecules (BCL2, BCL-XL, MCL1) and the pro-apoptotic molecule BIM were evaluated by Western blot in cetuximab-treated PDOs. BIM was consistently upregulated by cetuximab in all tested models. Increased BIM was sequestered by the anti-apoptotic BCL-XL, suggesting that EGFR blockade may render mCRC residual cells dependent on BCL-XL to elude cell death and, consequently, more susceptible to its inhibition. We therefore challenged a panel of 14 PDOs with different BH3 mimetics currently in preclinical or clinical development and found that only the combination of cetuximab with BH3 mimetics against BCL-XL strongly reduced cell viability and induced apoptosis. BCL-XL blockade enhanced cetuximab efficacy also in mCRC patient-derived xenografts, which displayed more durable tumor regression that was maintained also after treatment discontinuation. Notably, the combination of cetuximab with BCL-XL inhibitors was effective in vivo in all tested models except for one, which was characterized by low basal expression of BIM. Mechanistically, unleashed apoptosis by combination therapy was due to cetuximab-induced BIM upregulation concomitantly with its displacement from BCL-XL by the BH3 mimetic. Overall, this study offers new insights into the molecular mechanisms assisting residual disease after cetuximab treatment in mCRC, providing hints to design rational strategies that may convert the cytostatic effect of cetuximab into a cytotoxic, fully apoptotic outcome. Basal expression of BIM could be exploited as a predictive biomarker to enrich for potential responders to the combination of cetuximab and BH3 mimetics. Citation Format: Simonetta Maria Leto, Irene Catalano, Martina Ferri, Valentina Vurchio, Francesca Cottino, Eugenia Rosalinda Zanella, Francesco Sassi, Giorgia Migliardi, Andrea Bertotti, Livio Trusolino. Exploiting patient-derived xenografts and organoids to tackle apoptotic dependencies in EGFR-inhibited colorectal tumours [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 3697.

Reckoning apigenin and kaempferol as a potential multi-targeted inhibitor of EGFR/HER2-MEK pathway of metastatic colorectal cancer identified using rigorous computational workflow.

In the past two decades, the treatment of metastatic colorectal cancer (mCRC) has been revolutionized as multiple cytotoxic, biological, and targeted drugs are being approved. Unfortunately, tumors treated with single targeted agents or therapeutics usually develop resistance. According to pathway-oriented screens, mCRC cells evade EGFR inhibition by HER2 amplification and/or activating Kras-MEK downstream signaling. Therefore, treating mCRC patients with dual EGFR/HER2 inhibitors, MEK inhibitors, or the combination of the two drugs envisaged to prevent the resistance development which eventually improves the overall survival rate. In the present study, we aimed to screen potential phytochemical lead compounds that could multi-target EGFR, HER2, and MEK1 (Mitogen-activated protein kinase kinase) using a computer-aided drug design approach that includes molecular docking, endpoint binding free energy calculation using MM-GBSA, ADMET, and molecular dynamics (MD) simulations. Docking studies revealed that, unlike all other ligands, apigenin and kaempferol exhibit the highest docking score against all three targets. Details of ADMET analysis, MM/GBSA, and MD simulations helped us to conclusively determine apigenin and kaempferol as potentially an inhibitor of EGFR, HER2, and MEK1 apigenin and kaempferol against mCRC at a systemic level. Additionally, both apigenin and kaempferol elicited antiangiogenic properties in a dose-dependent manner. Collectively, these findings provide the rationale for drug development aimed at preventing CRC rather than intercepting resistance.

Examination of the functional and diagnostic potential of methylation-sensitive enhancers in metastatic colorectal cancer.

TPS237 Background: Molecular characterisation of colorectal cancer (CRC) has demonstrated the regulatory role of epigenetic alterations, such as DNA methylation, in CRC tumorigenesis(1). Robust molecular profiling of CRC has the potential to provide critical diagnostic and prognostic information. The establishment of 4 consensus molecular subtypes (CMSs), developed initially for primary CRC tumours, resulted in the evolving framework for molecularly targeted interventions; the molecular genetic profile characterising mCRC is less well defined(3, 4). Previous pilot work identified a unique tumour-specific methylation sequence, at 376 sites within the DNA of mCRC cells(5). This study will assess whether this methylation sequence drives mCRC pathogenesis and underpins disease phenotype. The primary aim of this study is validation of the diagnostic utility of this novel enhancer signature and identification of key enhancers with the potential to direct targeted treatment development. Secondary aims include CRISPR knockdown library development targeting the enhancers in CRC cells in vitro, evaluating the impact of the knockdown on phenotype in vitro, and identifying the mechanisms by which methylation-sensitive enhancers regulate the mCRC phenotype. Methods: This is a retrospective, non-interventional, single-centre clinical study, including patients > 18 years, with (group A) de novo mCRC (n = 100), (group B) stage III colorectal cancer that subsequently relapsed (n = 100) or (group C) stage III colorectal cancer without radiological evidence of relapse at study enrolment (n = 100). Formalin-fixed, paraffin-embedded ( FFPE) tissue obtained at diagnosis will be analysed. Additionally, 50 stage III patients who have relapsed (group B) will have blood samples collected prospectively at time of consent to enable assessment for the presence of the methylation signature on ctDNA. The presence or absence of the novel methylation signature will be evaluated via the application of a targeted bisulfite sequencing panel consisting of the previously identified differentially methylated enhancer (DME) signature to both tissue and plasma samples; results will be correlated with conventional histological parameters, systemic therapy, and overall survival. Quantitative statistical analysis will be performed using SPSS with linear regression analysis for survival data. Ethical approval was obtained from the MMUH Institutional Review Board; reference 1/378/2188. This study is open and recruiting. Clinical trial information: 1/378/2188.

Synergistic potential of dual andrographolide and melatonin targeting of metastatic colon cancer cells: Using the Chou-Talalay combination index method

Colorectal cancer (CRC) mortality has diminished for decades due to new and improved treatment profiles. However, CRC still ranks as the third most diagnosed cancer in the US. Therefore, a new therapeutic approach is needed to overcome colospheroids inhibition and drug resistance. It is well documented that andrographolide (AGP) and melatonin (MLT) have anti-carcinogenic properties. Our goal was to evaluate their synergistic effects on metastatic colon cancer cells (mCRC) and colospheroids. HT-29 and HCT-15 mCRC cells were simultaneously treated with serial dilutions of AGP and MLT for 24, 48 and 72 h. Cell viability was monitored using the MTT assay. The Chou-Talalay method for drug combination is based on the median effect equation, providing a theoretical basis for the combination index and the isobologram equation. This allows quantitative determination of drug interactions using the CompuSyn software, where CI < 1, = 1, and >1 indicates synergistic, additive, and antagonistic effects respectively. Our results demonstrate that AGP and MLT in combination show synergism with CI values of 0.35293 and 0.34152 for HT-29 and HCT-15 respectively and a fractional inhibition of Fa = 0.50–0.90, as shown by the Fa-CI plot and isobologram. The synergism value was validated in colospheroids (HT-29-s and HCT-15-s) based on morphology, viability, and colony formation and in 5-FU drug resistant cell (HT-29R and HCT-116R) viability. The mechanism(s) of decreased cell viability are due to the induction of ER stress proteins and angiogenic inhibition. Our results provide rationale for using AGP in combination with MLT on mCRC.

Impact of Andrographolide and Melatonin Combinatorial Drug Therapy on Metastatic Colon Cancer Cells and Organoids.

Background:The death rate (the number of deaths per 100 000 people per year) of colorectal cancer (CRC) has been dropping since 1980 due to increased screening, lifestyle-related risk factors, and improved treatment options; however, CRC is the third leading cause of cancer-related deaths in men and women in the United States. Therefore, successful therapy for CRC is an unmet clinical need. This study aimed to investigate the impacts of andrographolide (AGP) and melatonin (MLT) on CRC and the underlying mechanism.Methods:To investigate AGP and MLT anticancer effects, a series of metastatic colon cancer cell lines (T84, Colo 205, HT-29, and DLD-1) were selected. In addition, a metastatic patient-derived organoid model (PDOD) was used to monitor the anticancer effects of AGP and MLT. A series of bioassays including 3D organoid cell culture, MTT, colony formation, western blotting, immunofluorescence, and quantitative polymerase chain reaction (qPCR) were performed.Results:The dual therapy significantly promotes CRC cell death, as compared with the normal cells. It also limits CRC colony formation and disrupts the PDOD membrane integrity along with decreased Ki-67 expression. A significantly higher cleaved caspase-3 and the endoplasmic reticulum (ER) stress proteins, IRE-1 and ATF-6 expression, by 48 hours were found. This combinatorial treatment increased reactive oxygen species (ROS) levels. Apoptosis signaling molecules BAX, XBP-1, and CHOP were significantly increased as determined by qPCR.Conclusions:These findings indicated that AGP and MLT associated ER stress-mediated apoptotic metastatic colorectal cancer (mCRC) cell death through the IRE-1/XBP-1/CHOP signaling pathway. This novel combination could be a potential therapeutic strategy for mCRC cells.

Hydrogen Sulfide-Evoked Intracellular Ca2+ Signals in Primary Cultures of Metastatic Colorectal Cancer Cells.

Simple SummaryColorectal cancer (CRC) is the most common type of gastrointestinal cancer and the third most predominant cancer in the world. CRC is potentially curable with surgical resection of the primary tumor. The clinical problem of colorectal cancer, however, is the spread and outgrowth of metastases, which are difficult to eradicate and lead to a patient’s death. The failure of conventional treatment to significantly improved outcomes in mCRC has prompted the search for alternative molecular targets with the goal of ameliorating the prognosis of these patients. The present investigation revealed that exogenous delivery of hydrogen sulfide (H2S) suppresses proliferation in metastatic colorectal cancer cells by inducing an increase in intracellular Ca2+ concentration. H2S was effective on metastatic, but not normal, cells. Therefore, we propose that exogenous administration of H2S to patients affected by metastatic colorectal carcinoma could represent a promising therapeutic alternative.Exogenous administration of hydrogen sulfide (H2S) is emerging as an alternative anticancer treatment. H2S-releasing compounds have been shown to exert a strong anticancer effect by suppressing proliferation and/or inducing apoptosis in several cancer cell types, including colorectal carcinoma (CRC). The mechanism whereby exogenous H2S affects CRC cell proliferation is yet to be clearly elucidated, but it could involve an increase in intracellular Ca2+ concentration ([Ca2+]i). Herein, we sought to assess for the first time whether (and how) sodium hydrosulfide (NaHS), one of the most widely employed H2S donors, induced intracellular Ca2+ signals in primary cultures of human metastatic CRC (mCRC) cells. We provided the evidence that NaHS induced extracellular Ca2+ entry in mCRC cells by activating the Ca2+-permeable channel Transient Receptor Potential Vanilloid 1 (TRPV1) followed by the Na+-dependent recruitment of the reverse-mode of the Na+/Ca2+ (NCX) exchanger. In agreement with these observations, TRPV1 protein was expressed and capsaicin, a selective TRPV1 agonist, induced Ca2+ influx by engaging both TRPV1 and NCX in mCRC cells. Finally, NaHS reduced mCRC cell proliferation, but did not promote apoptosis or aberrant mitochondrial depolarization. These data support the notion that exogenous administration of H2S may prevent mCRC cell proliferation through an increase in [Ca2+]i, which is triggered by TRPV1.

Abstract 550: Determining efficacy of drug combinations identified by unbiased high throughput screening for RAS-mutated colorectal cancer

Abstract Background: Metastatic colorectal cancer (mCRC) is the second leading cause of cancer death in the US. The response rate to current systemic therapies is ~50% and most patients die within 2.5 years from diagnosis of metastasis. With ~5% of patients with mCRC benefiting from immunotherapies, effective therapies for ~95% of patients with mCRC are urgently required. Greater than 50% of patients with mCRC harbor mutations in the oncogenic driver RAS (KRAS, NRAS). As targeting RAS directly is technically challenging for the majority of RAS mutated sites, efforts have been concentrated on targeting MEK, a downstream mediator of RAS. However, targeting MEK with single agent therapy is ineffective in patients with mCRC. Aims: We aimed to perform unbiased high throughput screening (HTS) to identify drugs that, when combined to MEK inhibitors, would enhance their efficacy. Methods: We performed HTS with CRC cells grown in 2D and 3D cell culture using the MEK inhibitor trametinib as a backbone, and two different compound libraries composed of drugs either approved by the FDA or in clinical trials; 1) the NCI oncology set V, and, 2) a custom clinical compound set. Using the Bliss model of synergy, multiple compounds were identified to be synergistic with trametinib. In vitro and in vivo validation of synergy for a specific drug combination, dasatinib with trametinib, were performed. Results: Our unbiased HTS studies demonstrated that combining the SRC inhibitor dasatinib with trametinib was synergistic in both, 2D and 3D, CRC cell culture. This combination was further validated by MTT and colony formation assays. RPPA and western blot analyses of markers for apoptosis and various cell proliferation markers demonstrated that targeting SRC with MEK inhibits cell proliferation and increases cell death in multiple CRC cell lines as compared to single agents. In vivo studies in mice using RAS mutated CRC PDXs and cell lines with these drugs at clinically relevant doses, however, failed to demonstrate significant tumor growth inhibition or tumor regression. Conclusions: Although our unbiased HTS and other in vitro validation studies demonstrated that combining dasatinib and trametinib can enhance the efficacy of MEK targeted therapy in RAS mutated mCRC cells, careful in vivo preclinical studies failed to demonstrate enhanced efficacy of combining these two drugs. These studies highlight the need for performing careful in vivo preclinical studies as a prerequisite to translate in vitro findings into clinical studies. Further, our studies emphasize the importance for performing in vivo studies using drug doses that reflect clinically achievable doses in humans to identify therapies, either single or in combination, that may finally benefit our patients. This study also exemplifies the importance of publishing negative data so that others can pursue alternative strategies. Citation Format: Fan Fan, Reid Powell, Jason Roszik, Rui Wang, Xiangcang Ye, Clifford Stephan, Lee Ellis, Rajat Bhattacharya. Determining efficacy of drug combinations identified by unbiased high throughput screening for RAS-mutated colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 550.

Metformin selectively inhibits metastatic colorectal cancer with the KRAS mutation by intracellular accumulation through silencing MATE1

Metastatic colorectal cancer (mCRC) patients have poor overall survival despite using irinotecan- or oxaliplatin-based chemotherapy combined with anti-EGFR (epidermal growth factor receptor) drugs, especially those with the oncogene mutation of KRAS Metformin has been reported as a potentially novel antitumor agent in many experiments, but its therapeutic activity is discrepant and controversial so far. Inspiringly, the median survival time for KRAS-mutation mCRC patients with diabetes on metformin is 37.8 mo longer than those treated with other hypoglycemic drugs in combination with standard systemic therapy. In contrast, metformin could not improve the survival of mCRC patients with wild-type KRAS Interestingly, metformin is preferentially accumulated in KRAS-mutation mCRC cells, but not wild-type ones, in both primary cell cultures and patient-derived xenografts, which is in agreement with its tremendous effect in KRAS-mutation mCRC. Mechanistically, the mutated KRAS oncoprotein hypermethylates and silences the expression of multidrug and toxic compound extrusion 1 (MATE1), a specific pump that expels metformin from the tumor cells by up-regulating DNA methyltransferase 1 (DNMT1). Our findings provide evidence that KRAS-mutation mCRC patients benefit from metformin treatment and targeting MATE1 may provide a strategy to improve the anticancer response of metformin.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Induces Intracellular Ca2+ Release through the Two-Pore Channel TPC1 in Metastatic Colorectal Cancer Cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) gates two-pore channels 1 and 2 (TPC1 and TPC2) to elicit endo-lysosomal (EL) Ca2+ release. NAADP-induced EL Ca2+ signals may be amplified by the endoplasmic reticulum (ER) through the Ca2+-induced Ca2+ release mechanism (CICR). Herein, we aimed at assessing for the first time the role of EL Ca2+ signaling in primary cultures of human metastatic colorectal carcinoma (mCRC) by exploiting Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide (GPN), and nigericin, which dissipates the ΔpH which drives Ca2+ refilling of acidic organelles, caused massive Ca2+ release in the presence of a functional inositol-1,4,5-trisphosphate (InsP3)-sensitive ER Ca2+ store. Liposomal delivery of NAADP induced a transient Ca2+ release that was reduced by GPN and NED-19, a selective TPC antagonist. Pharmacological and genetic manipulations revealed that the Ca2+ response to NAADP was triggered by TPC1, the most expressed TPC isoform in mCRC cells, and required ER-embedded InsP3 receptors. Finally, NED-19 and genetic silencing of TPC1 reduced fetal calf serum-induced Ca2+ signals, proliferation, and extracellular signal-regulated kinase and Akt phoshorylation in mCRC cells. These data demonstrate that NAADP-gated TPC1 could be regarded as a novel target for alternative therapies to treat mCRC.

Identification of Cross Talk between FoxM1 and RASSF1A as a Therapeutic Target of Colon Cancer.

Metastatic colorectal cancer (mCRC) is characterized by the expression of cellular oncogenes, the loss of tumor suppressor gene function. Therefore, identifying integrated signaling between onco-suppressor genes may facilitate the development of effective therapy for mCRC. To investigate these pathways we utilized cell lines and patient derived organoid models for analysis of gene/protein expression, gene silencing, overexpression, and immunohistochemical analyses. An inverse relationship in expression of oncogenic FoxM1 and tumor suppressor RASSF1A was observed in various stages of CRC. This inverse correlation was also observed in mCRC cells lines (T84, Colo 205) treated with Akt inhibitor. Inhibition of FoxM1 expression in mCRC cells as well as in our ex vivo model resulted in increased RASSF1A expression. Reduced levels of RASSF1A expression were found in normal cells (RWPE-1, HBEpc, MCF10A, EC) stimulated with exogenous VEGF165. Downregulation of FoxM1 also coincided with increased YAP phosphorylation, indicative of tumor suppression. Conversely, downregulation of RASSF1A coincided with FoxM1 overexpression. These studies have identified for the first time an integrated signaling pathway between FoxM1 and RASSF1A in mCRC progression, which may facilitate the development of novel therapeutic options for advanced colon cancer therapy.

Stim and Orai mediate constitutive Ca2+ entry and control endoplasmic reticulum Ca2+ refilling in primary cultures of colorectal carcinoma cells

Store-operated Ca2+ entry (SOCE) provides a major Ca2+ entry route in cancer cells. SOCE is mediated by the assembly of Stim and Orai proteins at endoplasmic reticulum (ER)-plasma membrane junctions upon depletion of the ER Ca2+ store. Additionally, Stim and Orai proteins underpin constitutive Ca2+ entry in a growing number of cancer cell types due to the partial depletion of their ER Ca2+ reservoir. Herein, we investigated for the first time the structure and function of SOCE in primary cultures of colorectal carcinoma (CRC) established from primary tumor (pCRC) and metastatic lesions (mCRC) of human subjects. Stim1-2 and Orai1-3 transcripts were equally expressed in pCRC and mCRC cells, although Stim1 and Orai3 proteins were up-regulated in mCRC cells. The Mn2+-quenching technique revealed that constitutive Ca2+ entry was significantly enhanced in pCRC cells and was inhibited by the pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3. The larger resting Ca2+ influx in pCRC was associated to their lower ER Ca2+ content as compared to mCRC cells. Pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 prevented ER-dependent Ca2+ release, thereby suggesting that constitutive SOCE maintains ER Ca2+ levels. Nevertheless, pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 did not affect CRC cell proliferation and migration. These data provide the first evidence that Stim and Orai proteins mediate constitutive Ca2+ entry and replenish ER with Ca2+ in primary cultures of CRC cells. However, SOCE is not a promising target to design alternative therapies for CRC.

Abstract 4899: Establishment and characterization of SN-38 resistant metastatic colorectal cancer cells (SW620/SN100) reveals an ABCG2 (BCRP) transport mediated resistance

Abstract Despite advances in anticancer drug research, chemotherapy options and response rates for metastatic colorectal cancer (mCRC) are poor. Irinotecan, a topoisomerase I inhibitor, is a commonly used chemotherapy against mCRC as a single agent, or in combination with other anti-cancer drugs such as fluorouracil and leucovorin. However, acquired resistance to chemotherapy, especially in metastatic patients, is a critical cause of treatment failure. Multi-drug resistance (MDR) in cancer is a major challenge to chemotherapy success. Mechanisms of MDR involve deliberate alteration in cellular expression, function, and/or responses of the cancer. One of the most notable mechanism of MDR is an increased cellular drug efflux through up-regulation of membrane ATP-binding cassette (ABC) transporters. To investigate a new and clinically relevant mechanism of MDR in mCRC cells, SN-38 (the potent and active metabolite of irinotecan) was regularly administered to SW620 cells with drug-free intervals to reflect a drug induced resistance to treatment. Through cytotoxicity and western blotting assays on the newly formed SW620/SN100 cells, we discovered ABCG2 (BCRP) to be the main mechanism of SN-38 resistance, with no alterations in the topoisomerase I, ABCB1 (P-gp) or ABCC1 (MRP1). These results confirmed our initial hypothesis, given SN-38 is a known substrate of ABCG2. This discovery of SN-38 induced efflux in mCRC is of a larger importance because this mechanism not only limits the efficacy of the chemotherapy, but also poses a threat to the efficacy of a variety of pharmacologically dissimilar drugs which are substrates of ABCG2. As treatments for mCRC continue to be investigated it is important to also continue investigating resistant mechanisms of these drugs. More discoveries of MDR mechanisms which are specific to cancer patient populations will offer superior diagnosis, proper treatment regimens and greater prognosis for patient success. Citation Format: Anna Maria Barbuti, Zhe-Sheng Chen. Establishment and characterization of SN-38 resistant metastatic colorectal cancer cells (SW620/SN100) reveals an ABCG2 (BCRP) transport mediated resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4899.

In Vitro Killing of Colorectal Carcinoma Cells by Autologous Activated NK Cells is Boosted by Anti-Epidermal Growth Factor Receptor-induced ADCC Regardless of RAS Mutation Status

Treatment of advanced metastatic colorectal cancer (mCRC) patients is associated with a poor prognosis and significant morbidity. Moreover, targeted therapies such as anti-epidermal growth factor receptor (EGFR) have no effect in metastatic patients with tumors harboring a mutation in the RAS gene. The failure of conventional treatment to improve outcomes in mCRC patients has prompted the development of adoptive immunotherapy approaches including natural killer (NK)-based therapies. In this study, after confirmation that patients' NK cells were not impaired in their cytotoxic activity, evaluated against long-term tumor cell lines, we evaluated their interactions with autologous mCRC cells. Molecular and phenotypical evaluation of mCRC cells, expanded in vitro from liver metastasis, showed that they expressed high levels of polio virus receptor and Nectin-2, whereas UL16-binding proteins were less expressed in all tumor samples evaluated. Two different patterns of MICA/B and HLA class I expression on the membrane of mCRC were documented; approximately half of mCRC patients expressed high levels of these molecules on the membrane surface, whereas, in the remaining, very low levels were documented. Resting NK cells were unable to display sizeable levels of cytotoxic activity against mCRC cells, whereas their cytotoxic activity was enhanced after overnight or 5-day incubation with IL-2 or IL-15. The susceptibility of NK-mediated mCRC lysis was further significantly enhanced after coating with cetuximab, irrespective of their RAS mutation and HLA class I expression. These data open perspectives for combined NK-based immunotherapy with anti-epidermal growth factor receptor antibodies in a cohort of mCRC patients with a poor prognosis refractory to conventional therapies.

Targeting Differentially Expressed UPR Mediators in Mucin‐rich Colorectal Cancers and Conventional Colorectal Adenocarcinomas

BackgroundDespite the improved 5‐year survival rate, colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States and is sub‐classified according to mucin content as mucin‐rich CRC (MCRC, ≥50% mucin content) or conventional colorectal adenocarcinoma (CCRA, &lt;50% mucin content). Ten percent of CRCs are considered mucin‐rich and are reported to be less sensitive to chemotherapy treatment than CCRAs. The unfolded protein response (UPR) is an adaptive response activated when unfolded proteins accumulate in the endoplasmic reticulum (ER). When the UPR is incapable of dealing with an increased protein load, it leads to apoptosis. Despite the increased protein load and UPR activation, tumor cells are known to maintain protein homeostasis and avoid apoptosis.ObjectivesWe hypothesized that the higher protein load in MCRC cells, initiated by the high demand of producing mucins, induces greater ER stress and activation of the UPR. Here, we aimed to identify UPR markers uniquely upregulated in their expression in MCRC versus CCRA patient biopsies. We also utilized CRC cell lines with similar mucin and UPR profiles as the biopsies, to target differentially regulated UPR markers for inhibition as potential targets for CRC therapy.ResultsOur data showed that when compared to CCRA samples, MCRC biopsies express significantly higher mRNAs for several UPR markers such as IRE1α, XBP1, and CHOP, which significantly correlate (p&lt;0.0001, p&lt;0.0001, p=0.0003, respectively) with MUC6 expression. In contrast, CCRA samples expressed higher levels of several protein disulfide isomerases (PDIs) including PDIA1 (p=0.0001), PDIA3 (p&lt;0.0001), PDIA5 (p&lt;0.0001), and PDIA17 (p=0.0025), that are known to be the downstream of canonical transducers of the UPR. We also identified CCRA (Caco2) and MCRC (LS174T) cell lines with similar UPR profiles to the biopsies. Our data indicate that cells from the MCRC cell line were less responsive to LOC14, a PDI inhibitor, whereas the CCRA cell line, which expressed higher levels of several PDIs, were less proliferative and had a greater decrease in total cell numbers following 6 days of treatment with LOC14. Accordingly, treatment with LOC14 inhibited one of the PDIs, PDIA3 in the CCRA cell, but not the MCRC cell line.ConclusionsThese novel results suggest that CCRAs and MCRCs have different signatures of UPR markers and CCRAs may be more responsive to PDI inhibitors of the UPR than MCRCs. Further characterization of the UPR profiles of CCRAs and MCRCs could aid in the development of future anti‐cancer therapies and targeting of UPR transducers and their downstream mediators.Support or Funding InformationNIH R01 HL122383 and Department of Pathology and Laboratory Medicine (UVM) Startup Fund (VA)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting inflammatory monocytes in human metastatic colorectal cancer.

605 Background: Colorectal cancer (CRC) is the most common gastrointestinal malignancy. 60% of CRC patients are diagnosed with metastatic CRC (mCRC) and the 5-year survival is &lt; 20%. CCR2+ inflammatory monocytes (IM) are recruited from the bone marrow to the tumor microenvironment by the CCL2/CCR2 chemokine axis. At the tumor, they become tumor associated macrophages (TAM) and play a crucial role in promoting tumor progression, metastasis, and chemoresistance. While the importance of IM has been shown in other malignancies, little is known about their role in mCRC. Methods: Flow cytometry was performed on human and murine PBMCs, adjacent normal tissue, and tumors. Qualitative RT-PCR, ELISA, and confocal microscopy were performed for CCL2 expression. T-cell suppression assays were performed using CD14+ IM isolated from patient PBMCs and liver metastasis (LM). A murine model of CRC LM was created by hemispleen injection of luciferase-labelled MC38 CRC cells. Mice were treated with a CCR2 inhibitor and/or FOLFOX. Results: Prior to liver resection, mCRC patients have a higher percentage of IM in the peripheral blood compared to healthy donors (p &lt; 0.0001), and on multivariate analysis elevated monocytes were prognostic of poor survival. We also found higher levels of CCL2 in the serum of mCRC patients (p &lt; 0.01). Additionally, there was increased expression of CCL2 in LM compared to uninvolved tissue (p &lt; 0.01), with the production of CCL2 localized to mCRC cells. FACS analysis showed CCR2+ TAM were elevated in LM compared to adjacent normal liver (p &lt; 0.05) with a paucity of effector T-cells. CD14+ TAMs isolated from mCRC inhibited T-cell proliferation, illustrating the immune suppressive phenotype of these cells. In a murine model of CRC LM, treating mice with a CCR2 inhibitor alone or in combination with FOLFOX chemotherapy resulted in decreased tumor burden. FACS analysis of treated tumors showed increased effector T-cells in mice treated with CCR2 inhibitor. Conclusions: IM are involved in the progression of mCRC. Targeting these immunosuppressive cells with a CCR2 inhibitor decreases tumor burden in a murine model. This represents a potential novel treatment for mCRC.