Parental HT29 Cells Research Articles

Novel insights into the ecDNA formation mechanism involving MSH3 in methotrexate‑resistant human colorectal cancer cells.

Extrachromosomal DNAs (ecDNAs), also known as double minutes (DMs), can induce a fast increase in gene copy numbers and promote the development of cancer, including drug resistance. MutS homolog 3 (MSH3), a key protein in mismatch repair, has been indicated to participate in the regulation of DNA double‑strand break (DSB) repair, which has been reported to be associated with the formation of ecDNAs. However, it remains unclear whether MSH3 can influence drug resistance via ecDNAs in cancer. In the present study, high MSH3 expression was observed in methotrexate (MTX)‑resistant HT29 cells [DM‑ and homogeneously staining region (HSR)‑containing cells] compared with parental HT29 cells. Additionally, decreased amounts of ecDNAs, HSRs and amplified genes locating on ecDNAs and HSRs were detected following depletion of MSH3 and this could be reversed by overexpressing MSH3 in DM‑containing cells. No corresponding changes were found in HSR‑containing cells. The present study further verified the involvement of MSH3‑regulated DNA DSB repair pathways in the formation of ecDNAs by detecting the expression of core proteins and pathway activity. Furthermore, expulsion of ecDNAs/HSRs was detected and increased frequencies of micronuclei/nuclear buds with dihydrofolate reductase (DHFR) signals were observed in MSH3‑depleted DM‑containing cells. Finally, changes in MSH3 expression could affect DHFR amplification‑derived DHFR expression and cell sensitivity to MTX, suggesting that MSH3 may influence cancer drug resistance by altering the amount of ecDNAs. In conclusion, the present study revealed a novel mechanism involving MSH3 in the regulation of ecDNAs by DSB repair, which will have clinical value in the treatment of ecDNA‑based drug resistance in cancer.

Abstract 414: Targeting lipid metabolism to improve efficacy of braf-targeted therapy in colorectal cancer

Abstract Background: Aberrant lipid metabolism is associated with poor prognosis in colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of lipogenesis, is overexpressed and a therapeutic target in CRC. Overexpression of CD36, a fatty acid translocase, plays a pro-tumorigenic role in CRC. BRAFV600E is the mutation occurring in about 10-15% of CRC cases. BRAF-targeted therapy is effective, but quickly developed resistance is an issue. Our preliminary data show that development of resistance to BRAF-targeted therapy is associated with an increase in expression of FASN, CD36, accumulation of triglycerides (TGs), and mitochondrial respiration. Therefore, our central hypothesis is that inhibition of lipid metabolism will sensitize CRC cells to BRAF inhibitors and overcome acquired resistance. Methods: We established HT29 cells and primary PT130 and PT2449pt cells resistant to PLX8394, a novel BRAF inhibitor. IC50 curves, PrestoBlue viability, CytoSelect™ 24-Well Cell Invasion, and Triglyceride Assays, Seahorse XF analysis, western blot, and confocal microscopy were used to evaluate differences between parental and resistant cells. RNA-seq and lipid analysis were used to evaluate changes in gene expression and lipids levels. Combination of PLX8394 and TVB3664 (FASN inhibitor) was tested on cell viability in parental and resistant cells. Results: PLX8394 resistant cells have a higher IC50, an increased proliferation, and invasion compared to parental cells. An increase in invasion of resistant cells is associated with a decrease in E-cadherin. RNA sequencing shows a significant increase in FASN expression. Western blot on resistant cells confirms upregulation of FASN, CD36, and other lipogenic markers. Consistently, resistant cells have an increase in levels of triglycerides as compared to parental cells. Seahorse XF Cell Mito Stress Test shows that resistant cells forgo the Warburg effect and instead rely more heavily on oxidative phosphorylation. To further support our hypothesis, FASN shRNA knockdown of FASN in parental HT29 cells is more susceptible to PLX8394 treatment compared to control. We also observed the synergetic effect of PLX8394 and TVB3664 treatment on cell viability in parental cells, but not in resistant cells. Conclusion: Our study demonstrates that resistance to BRAF inhibitors is associated with a significant increase in proliferation, metastasis, and upregulation of lipid metabolism. We show that combination of FASN and BRAF inhibitors has a combinational effect on inhibiting cell viability in parental but not resistant cells, suggesting that the addition of FASN inhibitor to the standard regimen for BRAFV600E mutation positive patients can improve efficacy of these therapies. Additional screening of lipid metabolism-targeted therapies in combination with standard BRAF regimens are needed to develop novel and more efficacious strategies for CRC patients with BRAF mutations. Citation Format: Mariah E. Geisen, Dang he, Chi Wang, Jill M. Kolesar, Yekaterina Zaytseva. Targeting lipid metabolism to improve efficacy of braf-targeted therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 414.

Expression of the methylcytosine dioxygenase ten-eleven translocation-2 and connexin 43 in inflammatory bowel disease and colorectal cancer.

Inflammatory bowel disease (IBD) constitutes a substantial risk factor for colorectal cancer. Connexin 43 (Cx43) is a protein that forms gap junction (GJ) complexes involved in intercellular communication, and its expression is altered under pathological conditions, such as IBD and cancer. Recent studies have implicated epigenetic processes modulating DNA methylation in the pathogenesis of diverse inflammatory and malignant diseases. The ten-eleven translocation-2 (TET-2) enzyme catalyzes the demethylation, hence, regulating the activity of various cancer-promoting and tumor-suppressor genes. To investigate Cx43 and TET-2 expression levels and presence of 5-hydroxymethylcytosine (5-hmC) marks under inflammatory conditions both in vitro and in vivo. TET-2 expression was evaluated in parental HT-29 cells and in HT-29 cells expressing low or high levels of Cx43, a putative tumor-suppressor gene whose expression varies in IBD and colorectal cancer, and which has been implicated in the inflammatory process and in tumor onset. The dextran sulfate sodium-induced colitis model was reproduced in BALB/c mice to evaluate the expression of TET-2 and Cx43 under inflammatory conditions in vivo. In addition, archived colon tissue sections from normal, IBD (ulcerative colitis), and sporadic colon adenocarcinoma patients were obtained and evaluated for the expression of TET-2 and Cx43. Expression levels were reported at the transcriptional level by quantitative real-time polymerase chain reaction, and at the translational level by Western blotting and immunofluorescence. Under inflammatory conditions, Cx43 and TET-2 expression levels increased compared to non-inflammatory conditions. TET-2 upregulation was more pronounced in Cx43-deficient cells. Moreover, colon tissue sections from normal, ulcerative colitis, and sporadic colon adenocarcinoma patients corroborated that Cx43 expression increased in IBD and decreased in adenocarcinoma, compared to tissues from non-IBD subjects. However, TET-2 expression and 5-hmC mark levels decreased in samples from patients with ulcerative colitis or cancer. Cx43 and TET-2 expression levels were also investigated in an experimental colitis mouse model. Interestingly, mice exposed to carbenoxolone (CBX), a GJ inhibitor, had upregulated TET-2 levels. Collectively, these results show that TET-2 levels and activity increased under inflammatory conditions, in cells downregulating gap junctional protein Cx43, and in colon tissues from mice exposed to CBX. These results suggest that TET-2 expression levels, as well as Cx43 expression levels, are modulated in models of intestinal inflammation. We hypothesize that TET-2 may demethylate genes involved in inflammation and tumorigenesis, such as Cx43, potentially contributing to intestinal inflammation and associated carcinogenesis.

MiR-494-3p mediates oxaliplatin resistance of colorectal cancer cells via PTEN/AKT pathway

Purpose: To unravel the influence of miR-494-3p on the insensitivity of colorectal cancer (CRC) cells to oxaliplatin.Methods: The mRNA level of miR-494-3p in oxaliplatin-resistant HT-29 cells was evaluated with reverse transcript-polymerase chain reaction (RT-PCR). The cells were treated with miR-494-3p suppressor or mimic, and then apoptotic changes were determined flow cytometrically. Resistancerelated gene expressions were measured using RT-PCR and western blotting. In addition, in vivo mouse experiments were conducted.Results: MiR-494-3p expression in oxaliplatin-resistant HT-29 cells was much higher than that in parental HT-29 cells, accompanied by increased levels of MRP, P-gp, and AKT phosphorylation (p-AKT), and decreased phosphatase and tensin homolog (PTEN) (p < 0.001). The miR-494-3p mimic suppressed oxaliplatin-induced parental HT-29 cell apoptosis, while miR-494-3p inhibitor promoted oxaliplatin-resistant HT-29 cell apoptosis and decreased the levels of p-AKT, MRP and P-gp, while upregulating PTEN (p < 0.001). Furthermore, AKT inhibitor had similar effects as miR-494-3p inhibitor (p < 0.001). Experiments using nude mice demonstrated that inhibition of miR-494-3p accentuated the sensitivity of oxaliplatin-resistant HT-29 cells to oxaliplatin (p < 0.05).Conclusion: Suppression of miR-494-3p attenuates oxaliplatin insensitivity to CRC cells via a mechanism which may involve PTEN/AKT pathway. Therefore, miR-494-3p may be an effective target for overcoming drug resistance of CRC.

Isolation Set-up and Characterization of cancer stem cells from HT29 colon cancer cell line

Abstract Background: Cancer stem cells (CSCs) have critical roles in tumor initiation, progression, metastasis, resistance to chemoradiotherapy, and recurrence in various cancers such as colon cancer. Therefore, extensive studies are required to understand the CSCs mechanism of action and design novel cancer therapies. Successful isolation and characterization of CSCs from tumor tissues or cancer cell lines provide the proper opportunity for these kinds of studies. This study aimed to isolate and characterize CSCs from HT29 colon cancer cell lines. Methods: Sphere formation assay was used to isolate CSCs from the HT29 cell line. The expression of four stemness genes, including LGR5, SOX2, c-Myc, and Oct4, was assessed by real-time PCR. The CSC markers, CD 44 and CD 24, were also evaluated and compared between the parental HT29 cell line and HT29-derived spheres by flow cytometry. Statistical analysis was performed using GraphPad Prism version 6.0 and the Mann-Whitney U test. Results:HT29-derived sphere cells were successfully formed in serum-free media. The proportion of CD 24 and CD 44-expressing cells and the expression of LGR5, SOX2, c-Myc, and Oct4 stemness genes were significantly higher in CSCs isolated from HT29 spheres compared with parental HT29 cells. Conclusion:Sphere formation assay is a proper method for the isolation of CSCs from the HT29 cell line. The stemness markers LGR5, SOX2, c-Myc, Oct4,CD 44,andCD 24are suitable for the characterization of these cells.

Scutellarin resensitizes oxaliplatin-resistant colorectal cancer cells to oxaliplatin treatment through inhibition of PKM2

Although oxaliplatin is an effective chemotherapeutic drug commonly used for colorectal cancer (CRC) treatment, drug resistance usually occurs during the long-term use of it. It is urgent to create strategies to reduce the resistance of CRC cells to oxaliplatin. Oxaliplatin-resistant CRC cells (OR-SW480 and OR-HT29) were acquired through long-term exposure of CRC cells to oxaliplatin. It was found that OR-SW480 and OR-HT29 cells exhibited obvious lower sensitivity and a higher metabolism rate of glucose compared to their parental SW480 and HT29 cells, respectively. However, combination with scutellarin significantly resensitized the OR-SW480 and OR-HT29 cells to oxaliplatin-induced cytotoxicity. Mechanically, overexpression of pyruvate kinase isoenzyme M2 (PKM2) was responsible for the resistance to oxaliplatin in OR-SW480 and OR-HT29. Combination with scutellarin was able to inhibit the PKM2 activity and thus reduced the production of adenosine triphosphate (ATP) to sensitize the oxaliplatin-induced mitochondrial apoptosis pathway in both OR-SW480 and OR-HT29 cells. It was indicated that scutellarin resensitizes oxaliplatin-resistant CRC cells to oxaliplatin treatment through inhibition of PKM2.

Abstract 6340: Re-sensitization of irinotecan/SN38 resistant colorectal cancer cells by SCO-101

Abstract Introduction: Annually, more than 1 million people are diagnosed with Colorectal Cancer (CRC) and more than 500.000 die from CRC. Only three types of chemotherapy are currently being used worldwide to treat CRC patients: 5FU, oxaliplatin and irinotecan. However, chemotherapy resistance is present or develops in the majority of metastatic CRC patients. Resistance to anti-cancer drugs represents the main cause of cancer-related deaths. Thus, re-sensitization of chemotherapy resistant cancer cells constitutes a highly unmet medical need. Materials and Methods: The drug, SCO-101, is an oral drug and has previously passed 4 Phase I clinical trials where it demonstrated excellent PK (T1/2=15 hours) and favourable safety profile. We used two pairs of parental (sensitive) and SN38-resistant (SN38 is the active metabilote of irinotecan) human colon cancer cell lines (HT29 and LoVo). Cell viability in vitro during 72h exposure to SCO-101 and/or SN38 was adressed by MTT assays. The level of the SN38 efflux transpoter, ABCG2/BCRP, was investigated in the presence or absence of SCO-101. Additionally, various flux assays were applied to investigate the effect of SCO-101 on ABCG2/BCRP activity. A kinase screen revealed that the kinase SRPK1 was inhibited by SCO-101 and the importance of SRPK1 activity in re-sensitization of SN38 resistant colon cancer cells was investigated by two different synthetic SRPK1 inhbitors. Results: In parental HT29 and LoVo cells, SCO-101 and SN38 each had a dose-dependent effect on cell viability and no combinational effects were observed between the drugs. When exposing SN38-resistant cells to the combination of SCO-101 and SN38, a significant inhibitory effect on cell viability compared to either drug alone was observed, suggesting an additive or synergistic effect between these two drugs with SCO-101 re-sensitizing the resistant cells to SN38. Flux assays with ABCG2 /BCRP substrates clearly demonstrated that SCO-101 inhibited the activity of ABCG2 and protein analysis further demonstrated that SCO-101 causes degradation of ABCG2/BCRP. Cell viability assays with two different SRPK1 inhibitors demonstrated that this kinase is involved in re-sensitization to SN38. Conclusion and future perspectives: These preclinical studies demonstrate that SCO-101 sensitizes SN38-resistant colon cancer cells to SN38 through two different mechanisms of actions e.g. inhibition of the SRPK1 kinase and degradation of the ABCG2/BCRP drug efflux pump. We are now preparing a clinical phase II study enrolling metastatic CRC patients, who have developed acquired resistance to irinotecan containing treatment. These patients will be re-exposed to the combination of irinotecan containing treatment and SCO-101. Primary end-points will be safety and objective response rate according to RECIST 1.1. Secondary end-points will be clinical benefit rate. The study has been approved by the Danish Medicines Agency. In conclusion, SCO-101 represents a unique drug with an innovative dual mechanism of action. Citation Format: Jan Stenvang, Sophie Ambjørner, Khwajanezrabodin Sedighi, Peter Michael Vestlev, Lasse Saaby, Birger Brodin, Nils Brunner. Re-sensitization of irinotecan/SN38 resistant colorectal cancer cells by SCO-101 [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 6340.

MiR-138 Suppresses the PDK1 Expression to Decrease the Oxaliplatin Resistance of Colorectal Cancer.

BackgroundOxaliplatin is one kind of platinum-based drug. It is effective and commonly used in the treatment of colorectal cancer (CRC). However, development of acquired drug resistance is still a big obstacle during the oxaliplatin therapy. It is urgent to take strategies to decrease the oxaliplatin resistance of CRC.Materials and MethodsOxaliplatin-resistant HT29 and SW480 (HT29/R and SW480/R) cells were acquired through long-term exposure to oxaliplatin by using the routine HT29 and SW480 cells. Relative glucose consumption, lactate generation and LDH activity were tested to evaluate the glycolysis of CRC cell lines. MTT assays were conducted to evaluate the differences of oxaliplatin sensitivity between HT29/R (SW480/R) cells and their parental HT29 (SW480) cells. Regulation of miR-138 on PDK1 was confirmed through qRT-PCR, Western blot and dual-luciferase reporter assays. Reactive oxygen species (ROS) levels were measured by flow cytometry.ResultsHT29/R and SW480/R cells exhibited higher glucose consumption, lactate production and LDH activity compared to their parental HT29 and SW480 cells. However, oxygen consumption rate (OCR) in HT29/R and SW480/R cells is lower than that in HT29 and SW480 cells, respectively. Results of MTT assays showed that treatment with miR-138 can increase the cytotoxicity of oxaliplatin to HT29/R and SW480/R cells. Research on mechanisms showed that PDK1 was the target of miR-138. Overexpression of miR-138 can inhibit the expression of PDK1, and thus increase the OCR of HT29/R and SW480/R cells. Under the treatment of oxaliplatin, the miR-138-overexpressed HT29/R and SW480/R cells generated more amount of ROS to get into the apoptosis process.ConclusionOverexpression of miR-138 suppressed the PDK1 expression to decrease the oxaliplatin resistance of CRC.

MiR-361 enhances sensitivity to 5-fluorouracil by targeting the FOXM1-ABCC5/10 signaling pathway in colorectal cancer.

Colorectal cancer (CRC) is one of most common malignancies worldwide. 5-fluorouracil (5-FU) is a mainstay of CRC treatment, particularly in patients with advanced stages of the disease; however, 5-FU-based chemotherapy is not always effective and may result in progression of the disease. The present study investigated several candidate microRNAs (miRs) in parental and 5-FU-resistant HCT116 and HT29 cells, and identified miR-361 as a novel regulator of chemosensitivity. Overexpression of miR-361 enhanced the 5-FU susceptibility of parental and resistant HCT116 and HT29 cells in vitro. Impaired colony formation capacity and increased cell apoptosis (as determined via flow cytometry) was observed in resistant HCT116 and HT29 cells. Furthermore, forkhead box M1 (FOXM1) was identified as a target gene of miR-361 using a dual-luciferase reporter assay, western blotting and reverse transcription-quantitative PCR. Additionally, FOXM1 knockdown improved the cytotoxicity of 5-FU in resistant CRC. ATP binding cassette subfamily C members 5 and 10 (ABCC5/10) were found to be downstream effectors of miR-361. In conclusion, miR-361 increased chemosensitivity, at least in part, via modulation of FOXM1-ABCC5/10. miR-361 may serve as a potential therapeutic target for patients with CRC.

Inhibition of PGE2/EP4 receptor signaling enhances oxaliplatin efficacy in resistant colon cancer cells through modulation of oxidative stress

The platinum-based chemotherapeutic agent, oxaliplatin, is used to treat advanced colorectal cancer (CRC). Unfortunately, nearly all patients acquire resistance to oxaliplatin after long-term use, limiting its therapeutic efficacy. Since COX-2 and PGE2 signaling can impact colon cancer cell proliferation and survival, we examined how this pathway was affected in an oxaliplatin resistant colon cancer cell line. PGE2 levels were significantly elevated in oxaliplatin-resistant HT29 cells (OXR) compared to naïve parental HT29 cells (PAR). This increase was associated with elevated COX-2 (17.9-fold; P = 0.008) and reduced 15-hydroxyprostaglandin dehydrogenase (2.9-fold; P < 0.0001) expression. RNAi knockdown of microsomal prostaglandin E synthase-1, the rate-limiting enzyme in PGE2 synthesis, sensitized OXR cells to oxaliplatin. Downstream effects of PGE2 in OXR cells were also examined. Selective inhibition of the EP4 PGE2 receptor by the small molecule inhibitor, L-161,982 enhanced oxaliplatin-induced apoptosis in OXR cells. L-161,982 also reduced expression of the colonic stem cell markers, CD133 and CD44, and inhibited tumor sphere formation. The accumulation of intracellular reactive oxygen species (ROS), a key component of oxaliplatin cytotoxicity, was significantly increased by EP4 inhibition (2.4 -fold; P < 0.0001). Overall, our findings uncover an important role for the COX-2/PGE2/EP4 signaling axis in oxaliplatin resistance via regulation of oxidative stress.

Arylamine N-acetyltransferase 1 protects against reactive oxygen species during glucose starvation: Role in the regulation of p53 stability.

Human arylamine N-acetyltransferase 1 (NAT1) has been associated with cancer cell growth and invasion, but the underlying molecular mechanisms remain unknown. NAT1 is located on the short arm of chromosome 8 (8p21), a region that is commonly deleted in colon cancer. Previously, it was reported that HT-29 colon cancer cells, which have a large deletion at 8p21-22, show marked morphological changes, increased E-cadherin expression and altered cell-cell contact inhibition following down-regulation of NAT1 with shRNA. By contrast, no effects on growth were observed in HeLa cells. In the present study, cellular changes following knockout of NAT1 with CRISPR/Cas9 in HT-29 and HeLa cells were compared in the presence and absence of glucose. Cell growth decreased in both cell-lines during glucose starvation, but it was enhanced in HT-29 cells following NAT1 deletion. This was due to an increase in ROS production that induced cell apoptosis. Both ROS production and cell death were prevented by the glutathione precursor N-acetylcysteine. NAT1 knockout also resulted in a loss of the gain-of-function p53 protein in HT-29 cells. When p53 expression was inhibited with siRNA in parental HT-29 cells, ROS production and apoptosis increased to levels seen in the NAT1 knockout cells. The loss of p53 may explain the decreased colony formation and increased contact inhibition previously reported following NAT1 down-regulation in these cells. In conclusion, NAT1 is important in maintaining intracellular ROS, especially during glucose starvation, by stabilizing gain-of-function p53 in HT-29 cells. These results suggest that NAT1 may be a novel target to decrease intracellular gain-of -function p53.

LIMK/cofilin pathway and Slingshot are implicated in human colorectal cancer progression and chemoresistance.

Cofilin phospho-regulation is important for actin filament turnover and is implicated in cancer. Phosphorylation of cofilin is mediated by LIM kinases (LIMKs) and dephosphorylation by Slingshot phosphatases (SSH). LIMKs and SSH promote cancer cell invasion and metastasis and represent novel anti-cancer targets. However, little is known regarding LIMK/cofilin and SSH in human colorectal cancer (CRC). In this study, we aimed to address their expression and significance in human CRC. We evaluated expression of non-phosphorylated (active) and phosphorylated cofilin, LIMK1, LIMK2, and SSH1 by immunohistochemistry in 143 human CRC samples in relation to clinicopathologic parameters, response of metastatic disease to chemotherapy, and epithelial-mesenchymal transition (EMT) markers β-catenin, E-cadherin, and ZEB. We show that active cofilin, LIMK1, LIMK2, and SSH1 are overexpressed in human CRC and are associated with tumor progression parameters. SSH1 is an independent predictor of lymph node metastasis by multivariate analysis. LIMK1 and SSH1 expression is also higher in non-responders to chemotherapy, and SSH1 is shown by multivariate analysis to independently predict response of metastatic disease to chemotherapy. Active cofilin, LIMK1, LIMK2, and SSH1 also correlated with the EMT markers examined. In addition, immunofluorescence analysis showed increased expression of active cofilin, LIMK1, LIMK2, and SSH1 in HT29 colon cancer cells resistant to 5-fluorouracil compared to parental HT29 cells. Our results suggest that F-actin regulators LIMK/cofilin pathway and SSH1 are associated with CRC progression and chemoresistance representing promising tumor biomarkers and therapeutic targets in CRC.

Abstract 3608: Prostaglandin E-2 promotes resistance to oxaliplatin in colorectal cancer cells

Abstract Oxaliplatin, a platinum-based DNA crosslinking agent, has been used successfully as a first-line chemotherapeutic agent for the treatment of metastatic colorectal cancer (CRC). However, long-term treatment with oxaliplatin results in resistance in nearly all patients, limiting its therapeutic efficacy. Previous studies have shown that treatment of CRC cells with selective inhibitors of the enzyme Cyclooxygenase-2 (COX-2) leads to the development of drug resistance. One of the terminal products of the COX-2 pathway, prostaglandin E-2 (PGE2), is a pro-inflammatory lipid that has been implicated in colorectal tumorigenesis; however, it is unknown whether PGE2 plays a role in the development of drug resistance. In this study, we have begun to investigate the relationship between PGE2 and oxaliplatin resistance using the human CRC cell lines, HT29 and HCT116. Oxaliplatin-resistant HT29 cells (HT29-OXR) were generated by continuous exposure of parental HT29 cells to increasing concentrations of oxaliplatin over a period of 3 months. We have found that HT29-OXR cells produce significantly higher levels of PGE2 (3-fold increase, p&amp;lt;0.05) compared to the parental HT29 cell line. In addition, we have observed concomitantly elevated levels of the terminal PGE2 synthase, microsomal prostaglandin E synthase-1 (mPGES-1). Furthermore, we have demonstrated that the addition of exogenous PGE2 to both parental HT29 and HCT116 cells significantly (p&amp;lt;0.0001) decreased their sensitivity (IC50) to oxaliplatin, measured by the MTT cell viability assay. These findings suggest that PGE2 promotes resistance to oxaliplatin in CRC cells. FACS analysis showed that co-treatment of HT29 cells with exogenous PGE2 for 24 hours leaded to 27% reduction in oxaliplatin-induced Annexin V staining for early apoptosis, while exogenous PGE2 maintained G2/M arrest and simultaneously reduced the proportion of dead sub-G1 cells by approximately 80.4% in parental HCT116 cells. Our findings reveal a role for PGE2 in oxaliplatin resistance, possibly via regulation of apoptosis, and provide new data suggesting that targeting mPGES-1 may be a useful strategy for overcoming oxaliplatin resistance. Citation Format: Huakang Huang, Daniel W. Rosenberg. Prostaglandin E-2 promotes resistance to oxaliplatin in colorectal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3608. doi:10.1158/1538-7445.AM2015-3608

Streptococcus gallolyticus Pil3 Pilus Is Required for Adhesion to Colonic Mucus and for Colonization of Mouse Distal Colon.

Streptococcus gallolyticus is an increasing cause of bacteremia and infective endocarditis in the elderly. Several epidemiological studies have associated the presence of this bacterium with colorectal cancer. We have studied the interaction of S. gallolyticus with human colonic cells. S. gallolyticus strain UCN34, adhered better to mucus-producing cells such as HT-29-MTX than to the parental HT-29 cells. Attachment to colonic mucus is dependent on the pil3 pilus operon, which is heterogeneously expressed in the wild-type UCN34 population. We constructed a pil3 deletion mutant in a Pil3 overexpressing variant (Pil3+) and were able to demonstrate the role of Pil3 pilus in binding to colonic mucus. Importantly, we showed that pil3 deletion mutant was unable to colonize mice colon as compared to the isogenic Pil3+ variant. Our findings establish for the first time a murine model of intestinal colonization by S. gallolyticus.

Abstract A04: Changes in matrix metalloproteinase expression in SN-38 resistant colorectal cancer cells

Abstract The matrix metalloproteinases (MMPs) are a complex family of zinc-dependent proteolytic enzymes, which collectively are capable of degrading all components of the extracellular matrix (ECM). The ECM provides structure and support for normal tissues, and acts both as a barrier to and support for cancer cell invasion and metastasis. MMPs are therefore believed to play an important role in regulating the rate of cancer progression. However, the repeated failure of MMP inhibitors in clinical trials, as well as recent discoveries of novel MMP activities and localizations, has led to a re-evaluation of the roles of MMPs to the cancer process. In colorectal cancer (CRC) the outlook following disease relapse after surgery and initial chemotherapy is poor, due both to drug resistance and further dissemination of disease. We investigated the involvement of MMPs and related molecules in this context. We have generated a series of derivatives of the human CRC cell line HT29 that are resistant to SN-38, the active metabolite of the chemotherapeutic agent irinotecan. The principal representative cell line, HT29-S, has a slower proliferation rate than parental HT29 cells, and yet forms denser outgrowths in monolayer culture. The abundance and localization of several MMPs and tissue inhibitors of metalloproteinases (TIMPs) have been assessed in both HT29 and HT29-S cells by western blotting of cytosolic and nuclear protein fractions,and by immunofluorescence. HT29-S cells showed a lower expression of MMP1, MMP7, and MMP9, but also TIMP1 and TIMP2, compared to their parental counterparts, with some differences in cellular distribution. In particular, MMP1 gave a strong nuclear signal by both methods in HT29 cells, which was strongly reduced in HT29-S. A reduction in MMP expression will allow for an increased accumulation of ECM components. This may act through integrin receptors to activate survival pathways and contribute to the chemoresistance observed in HT29-S cells, and may also facilitate the spread of cancer cells by refining the ECM framework in advanced CRC. Citation Format: Spencer I T Berg, Murray J. Cutler, Jr., Jonathan Blay. Changes in matrix metalloproteinase expression in SN-38 resistant colorectal cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A04.

Abstract B64: Development of a novel strategy to overcome drug resistance by targeting ATP citrate lyase and de novo lipogenesis in colorectal cancer cells

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. Although the response rate to current systemic therapies is ∼50%, drug resistance develops in nearly all patients leading to 50,000 deaths each year. Overcoming drug resistance involves understanding the mechanisms by which cancer cells adapt to the genotoxic stress. Metabolic changes at levels of mitochondria function, glucose metabolism, and de novo synthesis of fatty acids frequently occurs in malignant cells and impacts tumor development and growth. Our laboratory established in vitro drug-resistant models of CRC cells by chronic exposure of HT29 cells to increasing doses of chemotherapeutic agents (oxaliplatin, 5-FU and SN38) over a period of 4–6 months. We recently reported a metabolic switch to the glycolytic phenotype due to mitochondria defects in the oxaliplatin-resistant CRC cells (HT29-OXR) (Zhou et al, Cancer Research, 2012). In this study, we tested the hypothesis that deregulation of de novo lipogenesis pathways plays an important role in chemoresistance of CRC cells. Methods: A previous study (Bose et al. Br J Ca, 2011) using unbiased proteomic profiling by mass spectroscopy (MS) was used to determine the proteomic signature of defective metabolic pathways in the oxaliplatin-resistant CRC cells (OXR cells). ATP-citrate-lyase (ACLy), the key enzyme of de novo lipogenesis pathway, was examined for protein levels and activation. The lipid content of resistant cells was examined by transmission electron microscope (TEM) and Oil Red staining. Transient knockdown of the ACLy protein by siRNA was used to study re-chemosensitization of the resistant cells. A small molecule inhibitor of ACLy was studied in combination with chemotherapeutic agents in resistant CRC cells for growth inhibitory (MTT assay) and cytotoxic effects (PARP cleavage and Annexin V staining). A cell free assay was used to test the potency of the ACLy inhibitor on ACLy activation. Results: Activated ACLy protein level (phosphorylation on S454) was demonstrated by Western blot analysis in the HT29-OXR and SN38-resistant (HT29-SNR) cells, but not in 5-FU-resistant (HT29-FUR) cells. The OxR cells showed a 2–3 fold increase in lipid droplets numbers (by TEM examination) and fatty acid content (by Oil Red staining) than the parental cells. Furthermore, transient knockdown of the ACLy protein by siRNA demonstrated a return to chemosensitization when cells were treated with oxaliplatin. IC50 values of the ACLy inhibitor for parental HT29 and HT29-OXR, -SNR and —FUR cells were ∼30μM. As a single agent, the ACLy inhibitor blocked phosphorylation of ACLy and induced apoptosis in a concentration-dependent manner in parental HT29 cells, and its resistant derivatives -OXR and -SNR cells. Combination of the ACLy inhibitor at concentrations sufficient to block ACLy phosphorylation with oxaliplatin and SN38 showed enhanced effects on growth inhibition (MTT) and apoptosis induction (PARP cleavage and Annexin V assay) in HT29 cells-OXR and -SNR cells. Conclusions: Chemoresistant CRC cells demonstrated: 1) increased de novo lipogenesis, 2) elevated levels of key lipogenesis enzymes ACLy, 3) dependence on ACLy activity for cell survival under cytotoxic stress. This metabolic switch likely contributes to the chemoresistant phenotype of CRC cells. Targeting an early step of de novo lipogenesis such as blocking ACLy activity may provide a novel strategy to overcome drug-resistance in CRC cells.

Abstract 3066: Effect of stable knockdown of NOX1 gene expression with siRNA in human colon cancer cells

Abstract NADPH oxidase (Nox)-dependent reactive oxygen species (ROS) play a critical role in cell signaling, cell proliferation, and mitogenesis. We have previously shown that silencing NOX1 gene expression with siRNA decreases cell growth and angiogenesis in human HT-29 colon cancer xenografts. To investigate the mechanism of growth inhibition, we studied cell cycle and signaling pathways. We found using RT-PCR and Western analysis that the expression of the INK4 inhibitors (p15, p18, p19), as well as CDK6, phospho-Cyclin E, Cyclin E, and Cyclin D1 were all decreased when NOX1 was decreased by &amp;gt;80%. We detected important changes in the Raf-MEK-ERK pathway that were associated with NOX1 knockdown. Our stable knock-out cells (clone 6A) showed lower phosphorylation of Rac1, c-Raf, MEK1/2, ERK1/2, p90RSK and CREB. To understand this observation, we studied the activity of Tyr and Ser/Threonine protein phosphatases (PP) in our stable siRNA containing cells (6A) compared to stable scrambled siRNA containing cells (SA) and parental HT-29 cells. PTP1B activity was elevated (100.4+4.4 pmol of phosphate/min/µg) in 6A cells compared to both SA (69.2+3.8) and parental (47.8+2.1) cell controls, P&amp;lt;0.05. Serine/Threonine protein phosphatases demonstrated a similar pattern; elevated in 6A cells (127.3+3.8 pmol of phosphate/min/µg) versus both SA cells (91.7+2.1) and the parental line (88.9+1.9), P&amp;lt;0.05. To demonstrate that protein phosphatases play a key role in the regulation of ERK-CREB signaling, we used a c-Raf antibody to immunoprecipitate the c-Raf-PPs complex, and detected PTP1B and PP2C proteins by Western analysis. These data indicate that siRNA-mediated gene silencing of Nox1 deactivated ERK-CREB signaling, and that increased activity of protein phosphatases, that occur as a consequence of decreased Nox-mediated ROS formation, might play role in modifying the growth enhancing effects of the MAP kinase pathway in HT-29 colon cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3066. doi:1538-7445.AM2012-3066

Abstract 4075: Chemoresistant colorectal cancer cells exhibit high glycolytic activity

Abstract Background: Overcoming drug resistance in colorectal cancer (CRC) requires an understanding the mechanisms by which cancer cells adapt to the genotoxic stress. We established oxaliplatin resistant CRC cells derived from HCT116 and HT29 cell lines (HCT116 OxR and HT29 OxR) (Yang AD et al, Clin Ca Res, 2007). The mechanisms by which these resistant clones reprogram their energy metabolism to gain a survival advantage remain unknown. In this study, we hypothesized that chemoresistant CRC cells would exhibit altered cellular metabolism in order to survive following chronic genotoxic stress. Methods: The expression level of the glycolytic enzymes Glut1, hexokinase II (HK2), LDHA and HIF1α were detected by Western Blotting. Glucose uptake and lactate production was calculated by measuring the concentration of glucose and lactate in the culture media. Intracellular ATP/ADP levels, oxygen consumption and mitochondria ATP production were quantified. An in vivo xenograft study was used to compare the growth rate and angiogenesis of parental and oxaliplatin-resistant cells. Results: Compared with the parental cells, both HT29-OxR and HCT116-OxR cells exhibited a metabolic phenotype with increased glycolysis as reflected by increased glucose uptake and lactate production. Glycolytic enzymes were upregulated in resistant cells including Glut1, HK2 and LDHA. HIF1α expression and VEGF levels in the conditioned media were increased in the resistant cells. The mitochondria of OxR cells demonstrated defective complex I/II substrate ATP production despite increased cellular oxygen consumption. Importantly, the OxR cells maintain higher levels of intracellular ATP and ATP/ADP ratio indicating a metabolic switch to glycolysis. In a tumor xenograft model, HT29 OxR cells grew significantly slower than parental HT29 cells. Interestingly, when OxR cells were mixed with parental HT29 cells (50:50 and 90:10), tumor growth and microvessel counts were significantly increased. Conclusion: Oxaliplatin-resistant cells demonstrated: 1) high aerobic glycolytic activity, 2) an increase in HIF1α and glycolytic enzymes, and 3) defective mitochondria function. This metabolic switch provides more ATP production and likely contributes to the chemoresistant phenotype. Altering energy metabolism may provide a novel strategy to overcome drug-resistance in CRC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4075. doi:10.1158/1538-7445.AM2011-4075

Abstract 596: ABCG2 is upregulated in SN-38 resistant colorectal cancer cells

Abstract Irinotecan, a member of the camptothecin chemotherapeutic drug family, is a first and second line therapy for patients with colorectal cancer. However, as with most chemotherapeutic therapies, patients’ tumors eventually develop drug resistance. Therefore, identifying mechanisms of chemotherapeutic resistance will allow for the development of additional molecular therapeutic targets. In this study SN-38, the highly insoluble active metabolite of irinotecan, was used to investigate corollary resistance to irinotecan. SN-38 resistant HT-29 cells were generated and compared to parental untreated HT-29 cells. Cells were characterized following treatment with either DMSO soulubilized SN-38 or IVECT encapsulated SN-38, a triblock copolymer micelle, in aqueous solution. The ATP-binding cassette transmembrane transporter protein, ABCG2, was found to have more than 40-fold expression in the SN-38 resistant HT-29 cells compared to the parental line. The increased expression of ABCG2 protein correlated with increased resistance to SN-38 in HT-29 cells and also with increased cell efflux. Cytotoxicity studies demonstrated that SN-38 resistant HT-29 cells were affected in a similar manner by IVECT polymer encapsulated SN-38 micelles and DMSO dissolved free SN-38. Cell cycle analysis showed that SN-38 resistant cells had an abrogated S phase arrest compared to parental cells. Cell treatment with free SN-38 was limited by its solubility in addition to DMSO toxicity. Further, IVECT polymer encapsulation improved SN-38 aqueous solubility allowing for increased concentration administration. Efflux proteins such as ABCG2 have been found to be upregulated in numerous types of cancers by several classes of chemotherapeutic drugs. Since transporter proteins have an important role in cell efflux, ABCG2 overexpression could serve as an additional target for pharmacological modulation. Finally, the increased solubility of SN-38 by IVECT polymer encapsulation allows for potential in vivo clinical applications of SN-38, a molecule that is 1000 times more active than its prodrug irinotecan. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 596. doi:10.1158/1538-7445.AM2011-596

RNAi-Mediated Knock-Down of Arylamine N-acetyltransferase-1 Expression Induces E-cadherin Up-Regulation and Cell-Cell Contact Growth Inhibition

Arylamine N-acetyltransferase-1 (NAT1) is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics.