Introduction

The large neutral amino acid transporter family (LAT1-4) is responsible for the cellular uptake of essential amino acids. LAT1, in particular, is dominantly expressed in cancer cells and attracts attention as a target for therapy. Previously, the tumor growth inhibitory effect of pan-LAT inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), has been reported in in vitro and in vivo therapeutic experiments. However, because BCH inhibits whole LAT family proteins including LAT1 and LAT2, it is expected that agents with high LAT1 specificity will be superior to BCH regarding fewer side effects. JPH203, a novel LAT1 inhibitor with high LAT1 specificity, was recently developed. We aimed to evaluate the efficacy and anti-tumor action mechanism of JPH203 in colorectal cancer (CRC).

Aims & Methods

LAT family gene expressions in public databases were analyzed using UCSC Xena, and LAT1 expression was evaluated using immunohistochemistry in surgically resected CRC. Moreover, we evaluated the growth inhibitory effect of JPH203 in several CRC cell lines. Furthermore, JPH203 treatment experiments were conducted using the allogeneic immune-responsive mouse model with abundant stroma, which was created by orthotopic transplantation of the mouse-derived CRC cell line CT26 and mesenchymal stem cells (MSCs). The anti-tumor effect was evaluated by comprehensive gene expression analysis using mouse tumor tissue-derived RNA and validated in mouse tumors, clinical specimens, and in vitro.

Results

Database analysis and immunohistochemistry for clinical specimens showed that LAT1 expression was cancer-dominant and correlated with prognosis. JPH203 showed high efficacy in in vitro and significantly reduced tumor size and metastasis in in vivo. RNA sequencing-based pathway analysis showed that not only tumor growth and amino acid metabolism pathways but also stromal activation-related pathways were suppressed. The inhibitory effect on tumor stroma was validated by vimentin immunostaining in clinical specimens, co-culture assay with CT26 and MSCs in vitro, and E-cadherin and vimentin fluorescent double staining in in vivo.

Conclusion

LAT1 expression in CRC plays a significant role in tumor progression, and JPH203 may inhibit CRC progression and tumor stroma. The stromal inhibitory effect of JPH203 may be beneficial in considering its application to other stroma-abundant carcinomas and its use combined with other drugs.

Introduction

Wee1 negatively regulates the G2/M checkpoint of the cell cycle, inhibiting cells with DNA damage from entering mitosis and inducing apoptosis. Although efficacy of Wee1 inhibitors as single agents or in combination with other agents has been reported in a variety of carcinomas and the association between Wee1 inhibitors and tumor immunity has been reported, the effect of Wee1 inhibitors on the tumor immune microenvironment in colorectal cancer (CRC) has not been clarified. Since p53 and KRAS are involved in cell cycle regulation at the G1/S checkpoint, mutations in them may increase dependence on the G2/M checkpoint for DNA repair, making Wee1 inhibitors more effective.

Aims & Methods

This study aimed to clarify the association between Wee1 expression and clinicopathological features in CRC and the effect of Wee1 inhibitors on the tumor immune microenvironment in CRC. Study 1: We performed immunostaining on 152 CRC cases surgically resected at our hospital between May 2013 and February 2015, and classified them into positive and negative groups according to the degree of Wee1 expression and investigated the association between Wee1 expression and clinicopathological features. Study 2: We measured Wee1 expression levels by Realtime PCR on 10 human-derived CRC cell lines and 2 mouse-derived CRC cell lines. We examined the effect of Wee1 inhibitor (AZD1775) on cell proliferation using Timelapse system. We examined the effect of Wee1 inhibitor on cell cycle using Cell Cycle Analysis Kit. We examined the effects of Wee1 inhibitor on apoptosis induction were examined using the Apoptosis Detection Kit. Study 3: We created the CRC orthotopic transplantation mouse model by transplanting the CRC cell line MC38 derived from C57BL/6 mice into the cecum of mice, comparing the tumor sizes of the control and treatment groups treated with Wee1 inhibitor for 21 days, and evaluating the effect of Wee1 inhibitor on the tumor immune microenvironment by immunostaining and RNA sequencing of mice tumor specimens.

Results

Study 1: No significant association was found between Wee1 expression and clinicopathological features or prognosis. However, overall survival rate was significantly higher in Wee1 positive and KRAS-mutant patients compared to Wee1 negative and KRAS wild-type patients. Study 2: Real time PCR showed that Wee1 expression was higher in most CRC cell lines than in normal colon mucosa. Wee1 inhibitor attenuated cell proliferation in a concentration-dependent manner in all CRC cell lines, and the effect was independent on Wee1 expression level. Cell cycle analysis showed that the percentage of cells in G2/M phase increased after exposure to Wee1 inhibitor, especially in cell lines with both p53 and KRAS mutations. In addition, the number of apoptotic cells increased after exposure to Wee1 inhibitor, but in the cell lines with KRAS mutation and p53 wild type, the number of apoptotic cells did not increase. Study 3: In the orthotopic transplanted mouse model, tumor volume was significantly smaller in the treated group compared to the control group, and immunohistological examination and RNA sequencing of mice tumor specimens showed enhanced tumor immunity and suppressed stromal reaction.

Conclusion

Wee1 inhibitor attenuated CRC cell proliferation through modulating cell cycle, especially in cell lines with p53 mutation and attenuated tumor growth via enhancement of tumor immune response and suppression of stromal reaction in orthotopic transplanted mouse model. Wee1 inhibitor AZD1775 could be the promising treatment option for CRC.

Introduction

Colorectal cancer (CRC) is considered to develop through interaction of driver mutations such as APC and KRAS mutations with tumor microenvironment (TME) and inflammatory factors. Although chronic inflammation is widely recognized as a risk factor for CRC, the effect of transient and mild inflammation on tumorigenesis remains unclear. This study aimed to evaluate the effects of mild inflammation on colorectal tumorigenesis using a genetically modified mouse model.

Aims & Methods

Three genetically engineered mouse models were used: CDX2P9.5-CreER^T2;Kras^LSL-G12D/+ （KRAS mut）, CDX2P9.5-CreER^T2;Apc^flox/+（APC mut）, and CDX2P9.5-CreER^T2;Apc^flox/+;Kras^LSL-G12D/+（APCKRAS mut）. Each model was examined with and without a 5-day administration of 1.5% Dextran Sulfate Sodium (DSS). All the mice were treated with tamoxifen at 6 weeks of age, and DSS-treated groups were administered DSS at 8 weeks; subsequently, all the mice were sacrificed at 20 weeks. Location, number, size, and histological features of the tumors were analyzed. Immunofluorescence staining (F4/80, CD163, CD4, FoxP3, CD8, and Ki67) was performed to investigate changes in the TME.

Results

In the absence of DSS, no tumors were observed in either KRAS mut or APC mut mice. However, in APCKRAS mut mice, small tumors developed in the proximal colon. Upon DSS administration, no tumors were detected in KRAS mut mice, whereas APC mut and APCKRAS mut mice exhibited multiple tumors throughout the colon. Moreover, in APCKRAS mut mice, the tumors in the proximal colon were significantly more numerous and larger than those in DSS-untreated mice (number: 6.83±1.33 vs 2.14±1.21 [P<0.01], size: 2.14±0.38mm vs 2.83±0.41mm [P=0.01] ).Immunofluorescence staining revealed no substantial differences in the proximal colon tumors of APCKRAS mut mice between DSS-treated and -untreated groups. However, when APCKRAS mut and APC mut mice were compared, the former exhibited an increase in Tregs and M2 macrophages, indicating the formation of an immunosuppressive TME.

Conclusion

This study demonstrates that even mild inflammation can promote colorectal tumorigenesis in APC muttation mice.

Introduction

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality and morbidity worldwide. Its tumor microenvironment is composed of a complex interplay of various monocytes, dendritic cells, macrophages and T-cells. Among them, there are various subtypes of T-cells, each of which plays a very important role and has been reported to be associated with prognosis in various cancers. In CRC, there are many reports on the association between CD8+ T-cells and Foxp3+ T-cells and prognosis. However, these are all reports mainly focused on advanced cancers. No studies on T-cells focused on early-stage CRC have been reported to date. Among early-stage CRC, submucosal deep invasive (T1b) CRC has a lymph node metastasis rate of about 10%. Although submucosal invasion depth, vascular invasion, histologic grade, or budding grade are known to be pathological risk factors of lymph node metastasis, there have been no reports evaluating the relationship between tumor microenvironment and lymph node metastasis. Therefore, in this study, we aimed to investigate the characteristics of T-cells according to the degree of CRC invasion and the association between T-cells and the risk of lymph node metastasis in T1b CRC.

Aims & Methods

Initially, 60 patients with colorectal neoplasia [20 with intramucosal neoplasia (IM group); 20 with submucosal invasive CRC (SM group); and 20 with advanced cancer (AD group)] treated at Hiroshima University Hospital from January 2011 to December 2015 were randomly selected. We examined changes in T-cells with tumor invasion. Next, 84 patients with T1b CRC who underwent initial surgical resection with lymph node dissection or additional surgical resection with lymph node dissection after endoscopic resection at Hiroshima University Hospital from March 2013 to July 2019 were selected. We examined the relationship between T-cells and lymph node metastasis in T1b CRC. For all specimens, the phenotype and number of T-cells were evaluated using the triple immunofluorescence method of CD4 (helper T-cell marker), CD8 (killer T-cell marker), and Foxp3 (regulatory T-cell marker). We counted T-cells by subtype in the center (CT) and the invasive front (IF) of the tumor and examined their association with lymph node metastasis.

Results

In the IM group, there was no significant difference in the number of any subtype between CT and IF, while in the SM and AD groups, all subtypes were more numerous in IF than CT. They were also predominantly more numerous as the degree of tumor invasion with IM, SM, and AD. The high number of Foxp3 in IF and the high ratio of Foxp3/CD4 and Foxp3/CD8 were positively correlated with lymph node metastasis.

Conclusion

High Foxp3 counts and high Foxp3/CD4 and high Foxp3/CD8 ratios in IF of T1b CRC may help predict lymph node metastasis.

Disclosure

The authors have no conflicts of interest or financial ties to disclose.

Introduction

Immune checkpoint blockade (ICB) for treatment of colorectal cancer (CRC) has been approved for clinical use since 2017. However, the approval of PD-L1 inhibitor for CRC has been encumbered by the lack of durable clinical benefits. Increasing evidence from other cancer types support that PD-L1 expression correlates to the efficacy of ICB and tumor PD-L1 is essential for ICB-mediated tumor cytotoxicity [1-3]. Concomitantly, many studies have also demonstrated the infrequent expression of PD-L1 in CRC cells [4, 5], thereby alluding to the possibility of low/absent PD-L1 expression as a primary obstacle to ICB efficacy.

Aims & Methods

Given the integral role of tumour microenvironment (TME) in shaping the tumour’s natural history, we sought to elucidate the role of tumour stroma in regulating CRC PD-L1 expression. To this end, we aim to rescue tumor PD-L1 and assess its utility in ICB-mediated anti-tumour immunity.
​To explore the role of different cell types in the TME on CRC PD-L1 expression, cocultures between major cell compartments of the TME and CRC cell lines were performed. Further various omics approaches were utilised to characterise the crosstalk between CRC and stromal cells that saw significant suppression of CRC PD-L1 expression.

Results

Myeloid cells are able to exert a significant suppression of PD-L1 expression in CRC and this suppression is regulated at a post-transcriptional level. The underlying mechanism is orchestrated by CRC metabolic shift from glycolysis to oxidative phosphorylation in response to harsh metabolic competition within the TME.

Conclusion

We have identified potential cellular compartment of the TME and mechanisms which may explain, in part, low/absent CRC PD-L1 expression. This can thus serve to evaluate appropriate external manipulations that can alleviate CRC PD-L1 suppression and explore the possibility of combinatory therapy with anti-PD-L1 ICB to improve its clinical efficacy and durability.

References

1. Herbst, R.S., et al., Atezolizumab for First-Line Treatment of PD-L1–Selected Patients with NSCLC. New England Journal of Medicine, 2020. 383(14): p. 1328-1339.
2. Cortes, J., et al., Pembrolizumab plus Chemotherapy in Advanced Triple-Negative Breast Cancer. New England Journal of Medicine, 2022. 387(3): p. 217-226.
3. Lau, J., et al., Tumour and host cell PD-L1 is required to mediate suppression of anti-tumour immunity in mice. Nature Communications, 2017. 8(1): p. 14572.
4. Llosa, N.J., et al., The Vigorous Immune Microenvironment of Microsatellite Instable Colon Cancer Is Balanced by Multiple Counter-Inhibitory Checkpoints. Cancer Discovery, 2015. 5(1): p. 43-51.
5. Taube, J.M., et al., Association of PD-1, PD-1 Ligands, and Other Features of the Tumor Immune Microenvironment with Response to Anti–PD-1 Therapy. Clinical Cancer Research, 2014. 20(19): p. 5064-5074.

Introduction

The widespread presence of N1-methyladenosine (m¹A) modifications controls RNA metabolism and is pivotal to fundamental biological processes. However, the understanding of RNA m¹A involved in cancer is still minimal. Here we reported for the first time the role and underlying mechanism of RNA m¹A methyltransferase TRMT61A in colorectal cancer (CRC), one of the most common causes of cancer-related deaths, and assessed its clinical significance.

Aims & Methods

RNA m¹A level was detected by liquid chromatography-mass spectrometry. Targeted CRISPR/Cas9 dropout screening was conducted to explore essential m¹A regulators. The expression and clinical implication of TRMT61A were investigated in three different human CRC cohorts (n=379). The role of TRMT61A was elucidated via in vitro CRC cell lines, patient-derived CRC organoids models, and in vivo CRC xenograft mouse models. The underlying mechanisms of TRMT61A were studied by integrative m¹A sequencing and RNA sequencing analyses. Nanoparticle-mediated siRNA delivery system was developed to deplete TRMT61A expression. Meanwhile, a potent TRMT61A inhibitor was identified by structure-based virtual screening. The therapeutic potential of targeting TRMT61A in vivo was assessed through different CRC mouse models.

Results

Higher RNA m¹A levels were observed in primary CRCs compared to paired adjacent normal tissues (n=24, P<.05), inferring the abnormal change of RNA m¹A profile during CRC development. Among different m¹A regulators, TRMT61A was identified as the top essential gene in different CRC cell lines using CRISPR/Cas9 screening. TRMT61A catalyzed RNA m¹A modification and was overexpressed in 81.3% (122/150) and 84.0% (42/50) of primary CRCs from two independent cohorts (both P<.001). Accordingly, TRMT61A mRNA expression was positively correlated with global RNA m¹A levels of CRCs (P<.05). Of note, high TRMT61A protein expression predicted poor survival of CRC patients (n=179, P<.05). Functionally, TRMT61A overexpression increased CRC cells proliferation, migration, and invasion, but suppressed apoptosis; while knockout of TRMT61A exerted opposite effects in vitro and significantly inhibited CRC xenograft growth, and lung and liver metastasis in vivo. Mechanistically, ONECUT2 was identified as a downstream target of TRMT61A by m¹A sequencing and RNA sequencing. Wild-type but not catalytically inactive TRMT61A increased the m¹A level of ONECUT2 to stabilize its mRNA, leading to elevated ONECUT2 expression that activated MAPK/ERK pathway. Notably, depletion of ONECUT2 or inhibition of MAPK/ERK abrogated the tumor-promoting role of TRMT61A, implying that TRMT61A induced ONECUT2-MAPK/ERK axis to facilitate CRC. Finally, we explored the therapeutic potential of targeting TRMT61A via genetic depletion or pharmacological inhibition of TRMT61A. We generated PEG-PLGA-based nanoparticles for delivery of TRMT61A-siRNA which posed a strong inhibitory effect against CRC growth in vivo. Meanwhile, our structure-based virtual screening of 100,100 compounds identified a highly potent and selective TRMT61A inhibitor PGG. PGG could directly bind to TRMT61A protein, as is demonstrated by thermal shift assay and drug affinity responsive targets stability assay. Treatment with PGG suppressed CRC xenograft growth and intestinal tumorigenesis of Apc^Min/+ mice, accompanied by decreased RNA m¹A levels.

Conclusion

We unravel for the first time the connections between TRMT61A-induced m¹A modification and CRC development. TRMT61A could serve as a prognostic marker and therapeutic target in CRC.

References

Zhang C, Jia G. Reversible RNA Modification N(1)-methyladenosine (m¹A) in mRNA and tRNA. Genomics Proteomics Bioinformatics 2018;16:155-161.
Shi H, Chai P, Jia R, et al. Novel insight into the regulatory roles of diverse RNA modifications: Re-defining the bridge between transcription and translation. Mol Cancer 2020;19:78.
Siegel RL, Miller KD, Goding Sauer A, et al. Colorectal cancer statistics, 2020. CA Cancer J Clin 2020;70:145-164.

Introduction

Colorectal cancer (CRC) is a leading global malignancy and a major cause of cancer-related mortality. Exosomes—small extracellular vesicles—mediate intercellular communication by transferring microRNAs, which regulate gene expression and influence tumor behavior. Elevated levels of miR-1290 have been observed in CRC patient blood and tumor tissues. However, the biological function of exosomal miR-1290 in CRC progression remains poorly understood.

Aims & Methods

Plasma and tissue samples from CRC patients and healthy individuals were collected with ethical approval. Exosomes were isolated from patient plasma and CRC cell line-conditioned media using ultracentrifugation and membrane affinity methods. In vitro, CRC cells were transfected with miR-1290 mimics or inhibitors to assess effects on proliferation (CCK-8, EdU assays), clonogenicity (colony formation), apoptosis (flow cytometry), and migration (transwell assay). Candidate miR-1290 targets were predicted bioinformatically, and CBFA2T3 was validated by dual-luciferase reporter assay. Stable CBFA2T3-overexpressing CRC cell lines were established to evaluate phenotypic rescue. In vivo, CRC xenografts in nude mice were treated with exosomes derived from miR-1290–overexpressing or control cells. Tumor growth and expression of Ki-67 and PCNA were analyzed.

Results

Plasma exosomal miR-1290 levels were lowest in healthy controls and progressively increased across advanced adenoma, early-stage CRC, and advanced CRC groups. Notably, miR-1290 distinguished advanced CRC from controls with an AUC of 0.77 and moderately distinguished early CRC (AUC = 0.69). Consistent with clinical findings, miR-1290 expression was significantly elevated in CRC tissues versus adjacent normal mucosa and in CRC cell lines compared to normal colonic epithelial cells. CRC cells also secreted exosomes enriched in miR-1290. In vitro, miR-1290 mimics enhanced CRC cell proliferation, DNA synthesis, and clonogenic potential, without significantly affecting apoptosis or migration. Bioinformatic analysis identified CBFA2T3 as a key target of miR-1290. Dual-luciferase assays confirmed direct binding of miR-1290 to the 3′-UTR of CBFA2T3, resulting in its downregulation. In CRC cells, miR-1290 upregulation—via mimics or exosomal transfer—decreased CBFA2T3 mRNA and protein levels. Restoration of CBFA2T3 expression significantly counteracted miR-1290-induced proliferation and colony formation. In vivo, nude mice injected with CRC cells and treated with miR-1290–enriched exosomes developed significantly larger tumors compared to controls. Tumors exhibited reduced CBFA2T3 levels and elevated expression of proliferation markers Ki-67 and PCNA, supporting the tumor-promoting role of exosomal miR-1290.

Conclusion

Exosomal miR-1290 correlates with CRC progression and shows promise as a non-invasive diagnostic biomarker. Functionally, it promotes CRC proliferation and tumor growth by targeting and downregulating the tumor suppressor CBFA2T3, highlighting its potential as a therapeutic target.