Colon Cancer Growth Research Articles

TRNA-Uridine Aminocarboxypropyltransferase DTW Domain Containing 2 Suppresses Colon Adenocarcinoma Progression.

DTW Domain Containing 2 (DTWD2) is a newly identified transfer RNA-uridine aminocarboxypropyltransferase. Dysregulated expression of DTWD1 has been reported in several malignancies, nevertheless, the role of DTWD2 in cancers remains completely unknown. Here, we aimed to initially investigate the expression and role of DTWD2 in colon adenocarcinoma. We first evaluated the transcription and mRNA levels of DTWD2 using data from The Cancer Genome Atlas. Besides, we tested its mRNA and protein expression in our enrolled retrospective cohort. Univariate and multivariate analyses were conducted to assess its prognostic value. Cellular experiments and xenografts were also performed to validate the role of DTWD2 in colon cancer progression. DTWD2 was downregulated in colon adenocarcinoma and associated with poor prognosis. Lymph node metastasis, distant metastasis, and advanced tumor stage are all characterized by lower DTWD2 levels. Furthermore, Cox regression analysis demonstrated that DTWD2 is a novel independent prognostic factor for colon cancer patients. Finally, cellular and xenograft data demonstrated that silencing DTWD2 significantly enhanced colon cancer growth. Low expression of DTWD2 may be a potential molecular marker for poor prognosis in colon cancer.

Suppression of colon cancer growth by berberine mediated by the intestinal microbiota and the suppression of DNA methyltransferases (DNMTs).

The purpose of this study was to demonstrate the regulatory effect of berberine (BBR) on the intestinal microbiota and related epigenetics during the inhibition of colon cancer cell growth in vitro and in vivo. We used a nude mouse xenograft model with HT29 colon cancer cells to establish and divide into a model group and BBR group. The mice were treated for four weeks, and HT29 cells in the BBR group were cultured for 48h. Cetuximab and the DNA transmethylase (DNMT) inhibitor 5-AZA-dC were added to HT29 cells. Tumour volume and weight were measured by hematoxylin-eosin (HE) staining for histopathological observation. Mouse faeces were collected, and the gut microbiota was analysed with 16S rDNA amplicons. The levels of cytokines in the supernatant of HT29 cells were measured by ELISA. A CCK-8 kit was used to examine the proliferation of HT29 cells, and RT‒PCR was used to measure the levels of c-Myc, DNMT1, DNMT3A, and DNMT3B. We found that BBR reduced the growth of colon cancer cells to a certain extent in vitro and in vivo, although the difference was not statistically significant compared with that in the model group. BBR significantly mediated the abundance, composition and metabolic functions of the intestinal microbial flora in mice with colon cancer. The effect of BBR on inflammatory cytokines, including IL-6, FGF, and PDGF, was not obvious, but BBR significantly downregulated IL-10 levels (P < 0.05) and reduced c-Myc, DNMT1, and DNMT3B levels (P < 0.05). Inhibiting DNMTs with 5-AZA-dC significantly suppressed the proliferation of HT29 cells, which was consistent with the effect of BBR. The inhibitory effect of berberine on colon cancer is related not only to the intestinal microbiota and its metabolic functions but also to the regulation of DNMTs.

A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway.

Human epidermal growth factor receptor 2 (HER2) is a protein that is overexpressed in some types of cancer, including breast and urothelial cancer. Here we found that HER2 was present in a portion of colon cancer patients, raising the possibility of using anti-HER2 therapy. RC48, a novel antibody-drug conjugate (ADC) comprising cytotoxic monomethyl auristatin E (MMAE) and an anti-HER2 antibody tethered via a linker, showed a comparable therapeutic effect in both HER2 low expressed (IHC2+/FISH- or IHC+) and high expressed urothelial cancer patients. In vitro studies using colon cancer cell lines showed that RC48 effectively impeded the proliferation of HER2-positive cells, indicating its potential as a treatment for HER2-positive colon cancer. Mechanism study showed that RC48 not only induces cell cycle arrest but also disrupts HER2-mediated restain of cGAS-STING signaling, potentially activating an immune response against the cancer cells. The administration of RC48 significantly reduced the growth of HER2-positive colon cancer and made HER2-positive colon cancer cells more susceptible to immunotherapy. The results of our study will contribute to determining the feasibility of RC48 as a therapeutic option for HER2-positive colon cancer.

Endogenous CCL21-Ser deficiency reduces B16–F10 melanoma growth by enhanced antitumor immunity

The chemokine CCL21 regulates immune and cancer cell migration through its receptor CCR7. The Ccl21a gene encodes the isoform CCL21-Ser, predominantly expressed in the thymic medulla and the secondary lymphoid tissues. This study examined the roles of CCL21-Ser in the antitumor immune response in Ccl21a-knockout (KO) mice. The Ccl21a-KO mice showed significantly decreased growth of B16–F10 and YUMM1.7 melanomas and increased growth of MC38 colon cancer, despite no significant difference in LLC lung cancer and EO771 breast cancer. The B16–F10 tumor in Ccl21a-KO mice showed melanoma-specific activated CD8+ T cell and NK cell infiltration and higher Treg counts than wild-type mice. B16–F10 tumors in Ccl21a-KO mice showed a reduction in the positive correlation between the ratio of regulatory T cells (Tregs) to activated CD8+ T cells and tumor weight. In Ccl21a-KO tumor, the intratumoral Tregs showed lower co-inhibitory receptors TIM-3 and TIGIT. Taken together, these results suggest that endogenous CCL21-Ser supports melanoma growth in vivo by maintaining Treg function and suppressing antitumor immunity by CD8+ T cells.

FRMD8 targets both CDK4 activation and RB degradation to suppress colon cancer growth

Cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (RB) are both important cell-cycle regulators that function in different scenarios. Here, we report that FERM domain-containing 8 (FRMD8) inhibits CDK4 activation and stabilizes RB, thereby causing cell-cycle arrest and inhibiting colorectal cancer (CRC) cell growth. FRMD8 interacts separately with CDK7 and CDK4, and it disrupts the interaction of CDK7 with CDK4, subsequently inhibiting CDK4 activation. FRMD8 competes with MDM2 to bind RB and attenuates MDM2-mediated RB degradation. Frmd8 deficiency in mice accelerates azoxymethane/dextran-sodium-sulfate-induced colorectal adenoma formation. The FRMD8 promoter is hypermethylated, and low expression of FRMD8 predicts poor prognosis in CRC patients. Further, we identify an LKCHE-containing FRMD8 peptide that blocks MDM2 binding to RB and stabilizes RB. Combined application of the CDK4 inhibitor and FRMD8 peptide leads to marked suppression of CRC cell growth. Therefore, using an LKCHE-containing peptide to interfere with the MDM2-RB interaction may have therapeutic value in CDK4/6 inhibitor-resistant patients.

Reactive oxygen species mediated apoptotic death of colon cancer cells: therapeutic potential of plant derived alkaloids.

Colorectal cancer (CRC) is one of the most deaths causing diseases worldwide. Several risk factors including hormones like insulin and insulin like growth factors (e.g., IGF-1) have been considered responsible for growth and progression of colon cancer. Though there is a huge advancement in the available screening as well as treatment techniques for CRC. There is no significant decrease in the mortality of cancer patients. Moreover, the current treatment approaches for CRC are associated with serious challenges like drug resistance and cancer re-growth. Given the severity of the disease, there is an urgent need for novel therapeutic agents with ideal characteristics. Several pieces of evidence suggested that natural products, specifically medicinal plants, and derived phytochemicals may serve as potential sources for novel drug discovery for various diseases including cancer. On the other hand, cancer cells like colon cancer require a high basal level of reactive oxygen species (ROS) to maintain its own cellular functions. However, excess production of intracellular ROS leads to cancer cell death via disturbing cellular redox homeostasis. Therefore, medicinal plants and derived phytocompounds that can enhance the intracellular ROS and induce apoptotic cell death in cancer cells via modulating various molecular targets including IGF-1 could be potential therapeutic agents. Alkaloids form a major class of such phytoconstituents that can play a key role in cancer prevention. Moreover, several preclinical and clinical studies have also evidenced that these compounds show potent anti-colon cancer effects and exhibit negligible toxicity towards the normal cells. Hence, the present evidence-based study aimed to provide an update on various alkaloids that have been reported to induce ROS-mediated apoptosis in colon cancer cells via targeting various cellular components including hormones and growth factors, which play a role in metastasis, angiogenesis, proliferation, and invasion. This study also provides an individual account on each such alkaloid that underwent clinical trials either alone or in combination with other clinical drugs. In addition, various classes of phytochemicals that induce ROS-mediated cell death in different kinds of cancers including colon cancer are discussed.

Retraction Note: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

Retraction Note: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

Somatic gain-of-function mutations in BUD13 promote oncogenesis by disrupting Fbw7 function.

Somatic mutations occurring on key enzymes are extensively studied and targeted therapies are developed with clinical promises. However, context-dependent enzyme function through distinct substrates complicated targeting a given enzyme. Here, we develop an algorithm to elucidate a new class of somatic mutations occurring on enzyme-recognizing motifs that cancer may hijack to facilitate tumorigenesis. We validate BUD13-R156C and -R230Q mutations evading RSK3-mediated phosphorylation with enhanced oncogenicity in promoting colon cancer growth. Further mechanistic studies reveal BUD13 as an endogenous Fbw7 inhibitor that stabilizes Fbw7 oncogenic substrates, while cancerous BUD13-R156C or -R230Q interferes with Fbw7Cul1 complex formation. We also find this BUD13 regulation plays a critical role in responding to mTOR inhibition, which can be used to guide therapy selections. We hope our studies reveal the landscape of enzyme-recognizing motif mutations with a publicly available resource and provide novel insights for somatic mutations cancer hijacks to promote tumorigenesis with the potential for patient stratification and cancer treatment.

Homeobox B9 Promotes Colon Cancer Progression by Targeting SRSF3.

Homeobox B9 (HOXB9) is one of the HOX family of transcription factors that are essential for cancer development and embryonic growth. However, the clinical importance and biological involvement of HOXB9 in colon cancer (CC) are not adequately understood. To investigate whether HOXB9 participates in the proliferation, invasion, and migration of CC. This study investigated the function and clinical significance of HOXB9 mRNA and protein expression in CC. Furthermore, overexpression and knockdown experiments of HOXB9 were developed to explore their effects on CC cell transwell and proliferation. Moreover, a molecular mechanism of HOXB9 regulate serine/arginine-rich splicing factor 3 (SRSF3) was explored. HOXB9 expression was higher in CC cells and tissues at both the mRNA and protein levels. Poor survival in CC patients was significantly connected with high HOXB9 expression, which was also strongly associated with the TNM stage and lymph node metastases. Furthermore, in vitro CC cell proliferation, transwell were markedly aided by HOXB9 overexpression. Contrarily, HOXB9 knockdown had the reverse result and inhibited the formation of xenograft tumors in naked mice. Gene set enrichment analysis (GSEA)revealed a correlation between high HOXB9 expression and spliceosomes. JASPAR and GEPIA2.0, in addition to CHIP and dual-luciferase reporting assays, confirmed that HOXB9 targets the promoter of SRSF3 to enhance its expression. We also found that SRSF3 knockdown eliminated HOXB9 from cell proliferation and transwell. We characterized the function and mechanism of HOXB9 in regulating colon cancer growth, suggesting a novel molecular approach for colon cancer-targeted therapy.

Eribulin mesylate exerts antitumor effects via CD103

ABSTRACT Eribulin mesylate (ERB) is a synthetic analog of halichondrin B, inhibiting tumor cell growth by disrupting microtubule function. Recently, anticancer drugs have been shown to not only act directly on tumor cells but also to exert antitumor effects by modifying the tumor environment. Although ERB has also been speculated to modify the tumor microenvironment including the immune response to tumors, the precise mechanism remains unclear. In our study, ERB suppressed the tumor growth of MC38 colon cancer in wildtype mice, whereas ERB failed to inhibit the tumor growth in Rag1-deficient mice which lack both B and T cells. Moreover, depletion of either CD4+ or CD8+ T cells abrogated the antitumor effect of ERB, indicating that both CD4+ and CD8+ T cells play an important role in ERB-induced antitumor effects. Furthermore, ERB treatment increased the number of tumor infiltrating lymphocytes (TILs) as well as the expression of activation markers (CD38 and CD69), immune checkpoint molecules (LAG3, TIGIT and Tim3) and cytotoxic molecules (granzyme B and perforin) in TILs. ERB upregulated E-cadherin expression in MC38. CD103 is a ligand of E-cadherin and induces T-cell activation. ERB increased the proportion of CD103+ cells in both CD4+ and CD8+ TILs. The ERB-induced antitumor effect with the increased TIL number and the increased expression of activation markers, inhibitory checkpoint molecules and cytotoxic molecules in TILs was abrogated in CD103-deficient mice. Collectively, these results suggest that ERB exerts antitumor effects by upregulation of E-cadherin expression in tumor cells and subsequent activation of CD103+ TILs.

Genetically predicted vitamin C levels significantly affect patient survival and immunotypes in multiple cancer types.

Recent observational studies and meta-analyses have shown that vitamin C reduces cancer incidence and mortality, but the underlying mechanisms remain unclear. We conducted a comprehensive pan-cancer analysis and biological validation in clinical samples and animal tumor xenografts to understand its prognostic value and association with immune characteristics in various cancers. We used the Cancer Genome Atlas gene expression data involving 5769 patients and 20 cancer types. Vitamin C index (VCI) was calculated using the expression of 11 genes known to genetically predict vitamin C levels, which were classified into high and low subgroups. The correlation between VCI and patient overall survival (OS), tumor mutational burden (TMB), microsatellite instability (MSI), and immune microenvironment was evaluated, using Kaplan-Meier analysis method and ESTIMATE (https://bioinformatics.mdanderson.org/estimate/). Clinical samples of breast cancer and normal tissues were used to validate the expression of VCI-related genes, and animal experiments were conducted to test the impact of vitamin C on colon cancer growth and immune cell infiltration. Significant changes in expression of VCI-predicted genes were observed in multiple cancer types, especially in breast cancer. There was a correlation of VCI with prognosis in all samples (adjusted hazard ratio [AHR] = 0.87; 95% confidence interval [CI] = 0.78-0.98; P = 0.02). The specific cancer types that exhibited significant correlation between VCI and OS included breast cancer (AHR = 0.14; 95% CI = 0.05-0.40; P < 0.01), head and neck squamous cell carcinoma (AHR = 0.20; 95% CI = 0.07-0.59; P < 0.01), kidney clear cell carcinoma (AHR = 0.66; 95% CI = 0.48-0.92; P = 0.01), and rectum adenocarcinoma (AHR = 0.01; 95% CI = 0.001-0.38; P = 0.02). Interestingly, VCI was correlated with altered immunotypes and associated with TMB and MSI negatively in colon and rectal adenocarcinoma (P < 0.001) but positively in lung squamous cell carcinoma (P < 0.05). In vivo study using mice bearing colon cancer xenografts demonstrated that vitamin C could inhibit tumor growth with significant impact on immune cell infiltration. VCI is significantly correlated with OS and immunotypes in multiple cancers, and vitamin C might have therapeutic potential in colon cancer.

Oncolytic vaccinia virus acts synergistically with anti-PD-L1 antibody to enhance the killing of colon cancer cells by CD8+ T cells

Both oncolytic vaccinia virus (OVV) and anti-PD-L1 antibody hold promise in cancer immunotherapy. Herein, we aimed to explore the possible synergistic effects of OVV and anti-PD-L1 on the growth and metastasis of colon cancer (CC) in mouse models. Microarray profiling of CC-related genes was first conducted. Expression of PD-L1 in CC tissues was predicted by TCGA and verified by flow cytometry and RT-qPCR. Then, mouse CC cell lines stably carrying luciferase MC38-luc and CT26-luc were infected with recombinant double-deleted vaccinia virus (vvDD) to evaluate the effect of vvDD on cell viability. The data indicated that PD-L1 was highly expressed in CC tissues and cells following vvDD infection. MC38-luc cells were inoculated into mice to construct CC-bearing mouse models, which were treated with vvDD or combined with anti-PD-L1, with tumor growth, metastasis, survival, and the immune environment analyzed. It was found that OVV combined with anti-PD-L1 antibody led to lower tumor burden and growth and higher survival rates than individual treatment in CC-bearing mice. In addition, this combination exerted a remote effect on the untreated subcutaneous tumors in the lateral abdomen, thus suppressing the tumor metastasis. Furthermore, combined therapy of OVV with anti-PD-L1 antibody activated CD8+ T cells, reduced exhaustion of CD8+ T cells, and enhanced their immune response, strengthening the killing of CC cells and inhibiting tumor growth and metastasis. In conclusion, our findings provide mechanistic insights into the action and efficacy of OVV as an immunomodulatory agent combined with the anti-PD-L1 antibody for the treatment of CC.

Differential Efficacy of Targeted Monoclonal Antibodies in Left-Sided Colon and Rectal Metastatic Cancers

BackgroundThe recommended first-line chemotherapy for RAS/BRAF wild-type metastatic colorectal cancer (mCRC) is bevacizumab (BEV)-containing therapy for right-sided colon cancer (R) and antiepidermal growth factor receptor antibody (anti-EGFR)-containing therapy for left-sided colon cancer (L) or rectal cancer (RE). However, anatomical or biological heterogeneity reportedly exists between L and RE. Therefore, we aimed to compare the efficacies of anti-EGFR and BEV therapies for L and RE, respectively. MethodsWe retrospectively reviewed 265 patients with KRAS (RAS)/BRAF wild-type mCRC treated with fluoropyrimidine-based doublet chemotherapy plus anti-EGFR or BEV as the first-line treatment at a single institution. They were divided into 3 groups: R, L, and RE. Overall survival (OS), progression-free survival (PFS), objective response rate, and conversion surgery rate were analyzed. ResultsForty-five patients had R (anti-EGFR/BEV: 6/39), 137 patients had L (45/92), and 83 patients had RE (25/58). In patients with R, both median (m) PFS and OS were superior with BEV therapy (mPFS, anti-EGFR vs. BEV: 8.7 vs. 13.0 months, hazard ratio [HR]: 3.90, P = .01; mOS, 17.1 vs. 33.9 months, HR: 1.54, P = .38). In patients with L, better mPFS and comparable mOS with anti-EGFR therapy were observed (mPFS, 20.0 vs. 13.4 months, HR: 0.68, P = .08; mOS, 44.8 vs. 36.0 months, HR: 0.87, P = .53), whereas, in patients with RE, comparable mPFS and worse mOS with anti-EGFR therapy were observed (mPFS, 17.2 vs. 17.8 months, HR: 1.08, P = .81; mOS, 29.1 vs. 42.2 months, HR: 1.53, P = .17). ConclusionsEfficacies of anti-EGFR and BEV therapies may differ between patients with L and RE.

Anti‑tumor effects of an aqueous extract of Eckloniacava in BALB/cKorl syngeneic mice using colon carcinoma CT26 cells.

Eckloniacava (E.cava) is well known as one of edible alga that contains various unique polyphenols. The anti‑tumor activity of an aqueous extract of E.cava (AEC) against colon carcinoma was evaluated by analyzing the alterations in tumor growth, histopathological structure and molecular mechanisms in CT26 tumor‑bearing BALB/cKorl syngeneic mice after administrating AEC for five weeks. AEC contained high total phenolic contents and demonstrated significant scavenging activity against 2,2‑diphenyl‑1‑picrylhydrazyl radicals. Marked anti‑tumor effects were demonstrated in the AEC‑treated CT26 cells. In the invivo syngeneic model, the AEC treatment decreased the volume and weight of CT26 tumors, and expanded the necrotic region in the hematoxylin and eosin stained tumor sections. The inhibitory effects of AEC on tumor growth were reflected by the increased level of apoptotic proteins, inhibition of cell proliferation, suppression of metastasis ability and increase in tumor‑suppressing activity in CT26 tumor‑bearing BALB/cKorl syngeneic mice. The potential function of phlorotannin (PT), one of the primary active compounds in AEC, was demonstrated by the increased cytotoxicity, apoptosis and suppression of cell proliferation in PT‑treated CT26 cells. Overall, the results of the present study provide novel scientific evidence that AEC can suppress the growth of CT26 colon cancer by activating apoptosis, suppressing cell proliferation, inhibiting cell migration and enhancing the tumor‑suppressing activity.

Design, synthesis, and biological activity of dual monoamine oxidase A and heat shock protein 90 inhibitors, N-Methylpropargylamine-conjugated 4-isopropylresorcinol for glioblastoma

Monoamine oxidase A (MAO A) and heat shock protein 90 (HSP90) inhibitors have been shown to decrease the progression of glioblastoma (GBM) and other cancers. In this study, a series of MAO A/HSP90 dual inhibitors were designed and synthesized in the hope to develop more effective treatment of GBM. Compounds 4-b and 4-c are conjugates of isopropylresorcinol (pharmacophore of HSP90 inhibitor) with the phenyl group of clorgyline (MAO A inhibitor) by a tertiary amide bond substituted with methyl (4-b) or ethyl (4-c) group, respectively. They inhibited MAO A activity, HSP90 binding, and the growth of both TMZ-sensitive and -resistant GBM cells. Western blots showed that they increased HSP70 expression indicating reduced function of HSP90, reduced HER2 and phospho-Akt expression similar to MAO A or HSP90 inhibitor itself. Both compounds decreased IFN-γ induced PD-L1 expression in GL26 cells, suggesting they can act as immune checkpoint inhibitor. Further, they reduced tumor growth in GL26 mouse model. NCI-60 analysis showed they also inhibited the growth of colon cancer, leukemia, non-small cell lung and other cancers. Taken together, this study demonstrates MAO A/HSP90 dual inhibitors 4-b and 4-c reduced the growth of GBM and other cancers, and they have potential to inhibit tumor immune escape.

GRIK5 stimulates colon cancer growth and metastasis through cAMP/PKA/CADM3 signaling.

Glutamate receptor, ionotropic, kainate 5 (GRIK5) is a member of glutamate receptors participating, and the kainate receptor family has been proved to regulate cell proliferation and transformation. Our study aimed at exploring the role of GRIK5 in colon tumor progression. Three hundred and ninety eightcolon cancer patients in The Cancer Genome Atlas Program (TCGA) data set and 26 clinical colon cancer patients were included for GRIK5 expression and prognosis analysis. GRIK5 overexpressed HCT116 and CT26 cell lines were established for cell proliferation and Transwell assay. Western blot analysis and immunostaining assay was conducted to evaluate the activation of activation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cell adhesion molecular 3 (CADM3) signaling in cell lines and tumor tissues. Subcutaneous CT26-bearing mice model was established to examine GRIK5-induced tumor growth and metastasis in vivo. Our study identified GRIK5 to be upregulated in patients with colon cancer, and higher GRIK5 levels correlated with the poor overall survival in patients. In vitro, GRIK5 overexpression markedly enhanced the proliferative properties and aggressive behaviors of colon cancer cells. Mechanistically, GRIK5 induced the activation of cAMP/PKA signaling, proceeded with augmented CADM3 expression, eventually resulting in sustained tumor growth.GRIK5 was a crucial scaffold in enabling colon cancer growth and metastasis, which offered a promising target for therapeutic manipulation of colon cancer.

A concise review on miRNAs as regulators of colon cancer stem cells and associated signalling pathways.

Despite recent therapy advances and a better understanding of colon cancer biology, it remains one of the major causes of death. The cancer stem cells,associated with the progression, metastasis, and recurrence ofcolon cancer, play a major role in promoting the development of tumour and are found to be chemo resistant. The stroma of the tumour, which makes up the bulk of the tumour mass, is composed of the tumour microenvironment. With the advent of theranostic and the development of personalised medicine, miRNAs are becoming increasingly important in the context of colon malignancies. A holistic understanding of the regulatory roles of miRNAs in cancer cells and cancer stem cells will allow us to design effective strategies to regulate miRNAs, which could lead to improved clinical translation and creating a potent colon cancer treatment strategy. In this review paper, we briefly discuss the history of miRNA as well as the mechanisms of miRNA and cancer stem cells that contribute to the tumour growth, apoptosis, and advancement of colon cancer. The usefulness of miRNA in colorectal cancer theranostic is further concisely reviewed. We conclude by holding a stance in addressing the prospects and possibilities for miRNA by the disclosure of recent theranostic approaches aimed at eradicating cancer stem cells and enhancing overall cancer treatment outcomes.

PTEN-induced kinase PINK1 supports colorectal cancer growth by regulating the labile iron pool

Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. However, the role of PINK1 in diseases where mitophagy has been purported to play a role, such as colorectal cancer, is unclear. Our results here demonstrate that higher PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth. We show that doxycycline-inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited proliferation, whereas disruption of other mitophagy receptors did not impact cell growth. We observed that PINK KD led to a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione. In addition, mitochondria are important hubs for the utilization of iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. We observed an increase in the iron storage protein ferritin and a decreased labile iron pool in the PINK1 KD cells, but total cellular iron or markers of iron starvation/overload were not affected. Finally, cellular iron storage and the labile iron pool are maintainedviaautophagic degradation of ferritin (ferritinophagy). We found overexpressing nuclear receptor coactivator 4, a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. These results indicate that PINK1 integrates mitophagy and ferritinophagy to regulate intracellular iron availability and is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells.

Abstract 4235: Obesity drives inflammation and metabolic reprogramming in an orthotopic mouse model of early colon cancer

Abstract Obesity is strongly associated with colon cancer incidence and, in animal models, promotes colon cancer growth and progression. Mounting evidence indicates that circulating growth factors and chronic inflammation are procancer factors likely mediating this relationship. While obesity blunts antitumor immunity in many cancer types, how obesity-driven inflammation and reduced antitumor immunity may cooperate to promote colon cancer progression is not yet clear. Here we used a murine syngeneic orthotopic transplantation model of colon cancer to profile changes driven by diet-induced obesity (DIO) in the immune and cancer cell compartments of the tumor using digital spatial profiling and RNA sequencing. 7-9-week-old male and female C57BL/6 mice remained on either a 10 kcal% fat diet (control, n=34) or a 60 kcal% fat diet (DIO, n=27) for at least 19 weeks. 700 Apc-null;KrasG12D/+;Trp53-null;Smad4-null;tdTomato (i.e., AKPS) organoids were transplanted into the colonic wall of control and DIO mice via colonoscopy-guided injection. Tumors were excised after four weeks of growth, an early time point to assess changes between diet groups independent of tumor size differences or weight loss associated with tumor growth. RNA sequencing was performed on RNA extracted from tumoral CD45+ and EpCAM+ cell fractions isolated using Milltenyi MicroBeads. DIO mice weighed more than controls (males: controls 37.0 g ± 5.1 vs. DIO 47.4 g ± 4.3; females: controls 26.4 g ± 3.7 vs. DIO 45.9 g ± 9.7) and had more mesenteric fat surrounding the colon (males: controls 388 mg ± 217 vs. DIO 577 mg ± 185; females: controls 201 mg ± 143 vs 1176 mg ± 589). Leptin, resistin, and plasminogen activator inhibitor-1 were significantly elevated in the serum of DIO mice relative to control mice. Tumor weight did not differ between groups. Gene set enrichment analysis demonstrated that tumor cells from DIO mice were significantly enriched for pathways related to inflammation (INFLAMMATORY_RESPONSE, IL6_JAK_STAT3_SIGNALING), glucose and lipid metabolism (GLYCOLYSIS, FATTY_ACID_METABOLISM), and proliferation (E2F_TARGETS, G2M_CHECKPOINT). Within the CD45+ transcriptomic profiles, Tumor Immune Estimation Resource (TIMER2.0) indicated an increase in M0- and M1-like macrophages and B cells in tumors from DIO compared with control mice. Analysis of whole tumor sections from DIO mice showed significantly reduced CD3+ cells by immunofluorescence and more Ki67+ CD45+ immune cells by digital spatial profiling relative to tumors from control mice. Taken together, obesity drives an inflammatory profile in both the immune and epithelial cell populations of AKPS murine tumors, which is associated with increased markers of proliferation. Future studies will address whether inflammatory signals from the mesenteric fat adjacent to the colon tumors may contribute to obesity-driven tumor inflammation, and thereby to colon cancer progression. Citation Format: Elaine M. Glenny, Babak Mirminachi, Tori L. McFarlane, Jatin Roper, Stephen D. Hursting. Obesity drives inflammation and metabolic reprogramming in an orthotopic mouse model of early colon 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 4235.

Abstract 1372: NUDT16L1 promotes colon cancer progression via inhibition of ferroptosis

Abstract Ferroptosis is an iron-dependent oxidative cell death with accumulation of lipid peroxidation and abnormal morphology of mitochondria. Although previous studies have shown that ferroptosis inducers have therapeutic potentials in several treatment-naive and drug-resistant cancer types, lacking systematic analysis of the ferroptosis sensitivity in different cancer types makes it difficult to know which cancer type is suitable for clinical usage and critical regulator determining the ferroptosis sensitivity during cancer progression is still not clear. In this study, colon cancer was identified as one of the ferroptosis-insensitive cancer types by dry and wet bench analyses. Importantly, our large-scale analysis also showed that NUDT16L1 was a novel ferroptosis repressor and overexpressed in colon cancer clinical specimens. Similar findings are also observed in the chemical-induced and genetic mouse models of colon cancer. Furthermore, NUDT16L1 loss-of-function and gain-of-function cell models and its conditional knock-in and knock-out mouse models are used to prove its crucial role in the development of colon cancer. In addition, loss of NUDT16L1 induced several key ferroptosis characteristics and impairment of mitochondrial functions while its restoration attenuated those phenomena in colon cancer cells. Mechanistically, our RNA-Seq and RIP-Seq analyses shown that NUDT16L1 directly interacted and positively regulated several crucial negative regulators of ferroptosis. More interestingly, loss of NUDT16L1 not only restores the sensitivity of ferroptosis inducer but also impairs the function of mitochondria. Finally, a specific NUDT16L1 inhibitor was shown to not only repress colon cancer growth in vitro and in vivo but also induce the ferroptosis in colon cancer. In conclusion, this is the first study to demonstrate that overexpression of NUDT16L1 promotes colon cancer progression by inhibition of ferroptosis and maintenance proper function of mitochondria. Furthermore, specific NUDT16L1 inhibitor might be a promising therapeutic strategy for colon cancer patient in the future. Citation Format: Yi-Syuan Lin, Ya-Chuan Tsai, Chia-Jung Li, Tzu-Tang Wei, Ya-Na Wu, Shang-Rung Wu, Shin-Chin Lin, Shaw-Jenq Tsai, Shih-Chieh Lin. NUDT16L1 promotes colon cancer progression via inhibition of ferroptosis [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 1372.