Society: AGA

Introduction & Aim:
Metachronous colorectal neoplasm (meta-CRN) may occur after colonoscopic polypectomy and subjects with metabolic derangement, such as metabolic syndrome and non-alcoholic liver disease (NAFLD), are associated with a higher risk of meta-CRN. Although our previous work demonstrated that active exercise after polypectomy can reduce the risk of metachronous advanced colorectal neoplasm (meta-ACRN), how it acts in high-risk subjects is unclear. The aim of this study is to elucidate whether exercise is an effective and specific preventive strategy against meta-ACRN among subjects with metabolic derangements.

Material & Method:
We retrospectively analyzed 3,231 subjects who underwent screening (index) colonoscopy with polypectomy and subsequent surveillance colonoscopy between January 2009 and December 2019. The baseline metabolic profiles of all participants were obtained at the date of index colonoscopy. All participants were also asked to complete a standard medical questionnaire before surveillance colonoscopy regarding their average exercise status after index colonoscopy. The risk of meta-ACRN after index colonoscopy in subjects with and without active exercise was analyzed by Kaplan-Meier (KM) analysis. Cox regression models are constructed for multivariate analysis.

Result
Of those 3,231 who were enrolled, the median age was 56 years old and 2247(65.5%) were men. Among the study cohort, 2450 (75.8%) subjects were classified as metabolic derangement (having at least one of the following factors: central obesity, elevated triglyceride, low HDL, hypertension and impaired fasting glucose) and 1134 (35.1%) subjects had NAFLD. A total of 1,365 (42.2%) subjects developed at least one meta-CRN including 166 (5.1%) subjects with meta-ACRN during a mean follow-up time of 3.4-year. After KM analysis, subjects adopting a more active exercise habitus (≥ 15 metabolic equivalent of tasks/week) after index polypectomy was associated a significantly reduced risk of meta-ACRN (log-rank p=0.02). In the multivariable analysis, the adjusted hazard ratio (aHR) of developing meta-ACRN after engaging active exercise after polypectomy was 0.52 (95% confidence interval (CI) = 0.63–0.75) for those with metabolic derangement and 0.24 (95% CI = 0.13–0.44) for those with NAFLD. On the other hand, the association between exercise status and meta-ACRN risk was non-significant in those with healthy metabolic status (aHR:0.65; 95% CI: 0.32-1.33)

Conclusion:
The study results confirmed the protective effect of active exercise after index polypectomy in preventing meta-ACRN and further demonstrated that such an effect was mediated by the subjects' metabolic status. For preventing meta-ACRN more specifically and efficiently, active exercise should be strongly advised after screening colonoscopy and polypectomy to subjects with metabolic derangements and NAFLD.

Introduction: The incidence of neuroendocrine neoplasms (NEN) has been reported to increase substantially over the last decades. The prevalence of NEN has to a less extent been reported.
Objective: We wanted to report the incidence and the prevalence of NEN in Norway from 1993 to 2021.
Methods: Incidence and the complete prevalence was investigated through the Cancer Registry of Norway, which covers the whole population and has been reported to be 99% complete. The neoplasms were classified into neuroendocrine tumors (NET) and neuroendocrine carcinomas (NEC).
Results: From 1993 to 2021 altogether 10.288 NET and 13.984 (1.758 outside the lungs) NECs were diagnosed. From 1993 to 2021 the incidence of NETs increased from 3,72 to 9,97 per 100.000 per year, corresponding to a 268% increase, p< 0,001. The largest increase in incidence for NETs was found for tumors originating from the pancreas (388%), lung (330%) and the small intestine (303%). The most common sites of origin for NETs in the last time-period, 2017-2021, were small intestine (23%), lung (19%) appendix (13%), pancreas (12%), rectum (5.6%) and colon (5.4%). In the last 5-years period, we observed a leveling out and even a fall in the incidence for several localizations (e.g. stomach, rectum, colon) compared with the previous 5-years period. For NEC there were no change in the incidence from 9,74 per 100.000 per year, to 9,95, p= 0,4, but there was an increase in the incidence for NECs originating from the skin (Merkel cell carcinoma) (289%), p<0.001 and from the GI-tract (212%) p=0.03. There were no changes in stage distribution for NETs and NECs. Metastatic disease at diagnosis was found in more than half of the NET patients and in more than 80% in the NEC patients. The prevalence for NENs increased 666% during the study period, NETs increased from 10.77 to 99,37 per 100.000 (923%), p<0,001. For NECs the increase was from 7,4 to 21,56 per 100.000 (291%), p<0,001. The largest increase was found for pancreatic (1155%), small intestinal (959 %) and lung (931%) NETs and NECs originating from the GI-tract (1880%) and skin (656%)
Conclusions: There were a substantially increase in incidence and prevalence of neuroendocrine neoplasm in Norway from 1993 to 2021.This is the first study to report complete prevalence of NENs for a whole nation.

Incidence of neuroendocrine tumors, 1993-2021

Incidence of neuroendocrine tumors, 1993-2021

Prevalence of neuroendocrine tumors, 1996-2021

Prevalence of neuroendocrine tumors, 1996-2021

Background: The inactivation of p53, a tumor suppressor, and the activation of the Ras oncogene are the most frequent genetic alterations in cancer. We have shown that a unique E. coli MazF-MazE toxin–antitoxin (TA) system, controlled by Ras and p53, delivered via adenoviruses can be used to selectively and effectively eradicate Ras mutated cancer cells (Shapira et al., 2021 oncogene). Because of their low immunogenicity and known advantage as ideal cell transporters, exosomes offer great promise as cancer immunotherapy agents. CD24 is a GPI-anchored protein that highly expressed in most of human malignancies and not normal cells.
Aim: Development of a drug delivery platform for effective and selective eradication of cancer cells while sparing normal cells based on the Trojan horse strategy and genetic signature.
Methods: Two delivery systems have been designed and developed; targeted lentivirus and exosomes (Fig.1). Trex293 cells were used to establish stable scFv-CD24-expressing clones, which were adapted to suspension culture in a chemically-defined media. Exosomes were purified by polymer precipitation method and characterized by several methods including nano-particle tracking analysis, Western blot and EXO-ELISA. CD24 stable transfected HCT116^-/-(P53 null) and HCT116^+/+ (WTP53) clones were established, and evaluated for CD24 and P53 expression. HCT116^+/+-CD24 cells were treated with exosomes loaded with GFP or MazF expressing plasmids and analyzed for GFP expression and cell viability.
Results: Targeted lentiviruses carrying the TA system efficiently shrink CRC-derived xenografts tumors in mice (Fig.2). Then, a new genetically encoded pseudotyping platform, based on exosomes, was developed. A homogenic and pure population of exosomes was purified from 3D culture of the highest expressing clone (TREx-scFv). Stable and high expression of CD24 were shown, by Western blot and flow cytometry, in both, HCT116^-/- and HCT116^+/+ cells. HCT116^+/+-CD24 cells treated with 5FU showed active P53. The ability of loading DNA into the purified exosomes and deliver them to the target cells were evaluated and showed high and intensive GFP expression. mazF was successfully loaded into the scFv targeted-exosomes and induced a massive cell death, in a dose-dependent manner.
Conclusions: By exploiting the P53 genetic status as well as the Ras pathway, an effective and safe treatment could be developed that targets tumor cells specifically while sparing normal tissues. The effectiveness of this out-of-the-box approach holds great promise for the treatment and management of cancer.

The Pseudotyping Approach to Load Exosomal Membranes

The Pseudotyping Approach to Load Exosomal Membranes

mazEF Loaded Lv-scFvs Effiently Inhibit Tumor Growth In Mice

mazEF Loaded Lv-scFvs Effiently Inhibit Tumor Growth In Mice

[Background & Aim] The number of patients with esophagogastric junction adenocarcinoma (EGJAC) is increasing along with the high incidence of metabolic syndrome (MetS). Previous studies demonstrated that a high fat diet (HFD) might cause MetS via gut dysbiosis and hypercytokinemia, and lead to colon cancer development, but the causal relationship between MetS and EGJAC remains unclear. Considering that gastric acid secretion may be preserved in EGJAC patients, we aimed to investigate whether local exposure to acidic bile salt (BS) promotes inflammation-associated carcinogenesis along with inter-organ communication with MetS-related dysbiosis. [Methods] First, we immunohistochemically investigated the carcinogenic role of reactive oxidative stress (ROS) using surgical specimens of 24 EGJAC patients. Second, we compared the effects of 15-week oral administration of standard diets/ HFD ± pH4BA/ pH7BA on tumor growth, gut barrier, and immune response using 20-weeks old Gan mice crossbreed with/without Nrf2-knockout mice. Third, we used human gastric cancer cell lines, MKN7 and NUGC4, and PMA-treated THP-1 to uncover the carcinogenic role of ROS and the Warburg effect by exposure to acidic BA. [Results] 1) Human study: The expressions of nitrotyrosine and 4-hydroxynoenal in the EGJACs with high infiltration of macrophages^Iba+ were higher than those in non-cancerous mucosa. 2) Animal study: Protruded tumors were observed around the junction between the forestomach and glandular stomach in all Gan mice. The ratio of tumor to the whole stomach, and the percentage of the Ki67- or TUNEL-positive cells in tumors with rich infiltration of macrophages^Iba+ in the HFD+pH4BA group were larger than those of the other groups. The expressions of Warburg effect-related genes, such as Hif1a, Glut1, Hk2, and Pkm2, and the production of lactate and 8-OHdG in the HFD+pH4BA group were higher than the others. As for gut barrier dysfunction, western blot analyses revealed a decrease of the ZO-1 expression in intestinal mucosa and an increase of the lipopolysaccharide expression in the liver in the HFD±pH4BA group. Moreover, tumor size, macrophage^Iba+ infiltration, and the expressions of 8-OHdG and the Warburg effect-related genes were decreased in the Nrf2-knockout Gan mice, compared to Gan mice. 3) in vitro study: In MKN7, the exposure to pH4BA increased ROS production and glucose uptake with induction of Hif1a and Glut1. The exposure with pH7BA, rather than pH4BA, to the PMA-treated THP1 induced TLR4mRNA and Nrf2mRNA. [Conclusion] We demonstrated that HFD might promote EGJAC via the Warburg effect by the ROS production due to the direct exposure of mucosa to acidic BA, as well as the migrated macrophage, which might be activated by the transudates through gut barrier dysfunction.

Background and aims: Metastasis accounts for the high mortality rates of colorectal cancer (CRC) patients. However, the molecular mechanism manipulating metastasis in CRC is still elusive. Here, we investigated the function of E74-like factor 4 (ELF4), a member of the ETS family, in facilitating CRC invasion and metastasis. Methods: ELF4 expression levels were analyzed by immunohistochemistry in two independent cohorts of human CRC tissues. The clinical significance of ELF4 was assessed by Kaplan-Meier analysis and Multivariate Cox proportional hazards model. The transcriptional regulation of fibroblast growth factor receptor 4 (FGFR4) and SRC proto-oncogene, non-receptor tyrosine kinase (SRC) by ELF4 were measured using serial deletion, site-directed mutagenesis, and chromatin immunoprecipitation assays. The potential role of FGFR4 and SRC in ELF4-mediated CRC invasion and metastasis were analyzed using Transwell assays and in vivo lung and liver metastasis models. Results: Increased expression of ELF4 has a positive correlation with distant metastasis, advanced AJCC stage, and poor outcomes in CRC patients. ELF4 expression was also an independent predictor for poor prognosis in CRC patients. Overexpression of ELF4 boosts CRC metastasis via transactivating its downstream target genes FGFR4 and SRC. Fibroblast growth factor 19 (FGF19), the ligand of FGFR4, upregulated ELF4 expression through the ERK1/2/SP1 axis, which forms a FGF19-ELF4-FGFR4 positive feedback loop. Clinically, ELF4 expression was positively correlated with FGF19, FGFR4 and SRC in human CRC specimens, and CRC patients who positively co-expressed FGF19/ELF4, ELF4/FGFR4, or ELF4/SRC exhibited the worst clinical outcomes. Furthermore, the combination treatment of FGFR4 inhibitor BLU-554 and SRC inhibitor KX2-391 dramatically suppressed ELF4-induced CRC metastasis. Conclusions: We demonstrated that FGF19-FGFR4 upregulated ELF4 expression through the ERK1/2/SP1 pathway, and ELF4 promotes CRC metastasis through upregulating FGFR4 and SRC expression, which formed a FGF19-ELF4-FGFR4 positive feedback loop. Targeting the FGF19-ELF4 pathway might represent a novel therapeutic strategy to inhibit CRC metastasis.

Figure 1. Elevated expression of ELF4 facilitates CRC metastasis and indicates poor prognosis in human CRC patients. (A) IHC images and scores of ELF4 expression in CRC tissues and adjacent nontumor tissues. (B) Kaplan-Meier analysis of the correlation of ELF4 expression with overall survival and recurrence. (C) ELF4 mRNA levels in CRC tissues and adjacent nontumor tissues. (D) IHC images and scores of ELF4 staining in primary CRC and paired metastatic CRC tissues. (E) Western blotting analysis. (F) Transwell assays. (G-J) ELF4 promotes CRC lung metastasis <i>in vivo</i>. (G) Bioluminescence images and mice lung metastasis incident rates. (H) Number of lung metastatic nodules. (I) Overall survival. (J) H&E staining of lung tissues. (K-N) ELF4 promotes CRC liver metastasis<i> in vivo. </i>(K) Bioluminescence images and mice liver metastatic rates. (L) Number of tumor foci on liver surface. (M) Overall survival. (N) H&E staining of liver tissues.

Figure 1. Elevated expression of ELF4 facilitates CRC metastasis and indicates poor prognosis in human CRC patients. (A) IHC images and scores of ELF4 expression in CRC tissues and adjacent nontumor tissues. (B) Kaplan-Meier analysis of the correlation of ELF4 expression with overall survival and recurrence. (C) ELF4 mRNA levels in CRC tissues and adjacent nontumor tissues. (D) IHC images and scores of ELF4 staining in primary CRC and paired metastatic CRC tissues. (E) Western blotting analysis. (F) Transwell assays. (G-J) ELF4 promotes CRC lung metastasis in vivo. (G) Bioluminescence images and mice lung metastasis incident rates. (H) Number of lung metastatic nodules. (I) Overall survival. (J) H&E staining of lung tissues. (K-N) ELF4 promotes CRC liver metastasis in vivo. (K) Bioluminescence images and mice liver metastatic rates. (L) Number of tumor foci on liver surface. (M) Overall survival. (N) H&E staining of liver tissues.

Figure 2. ELF4 facilitates CRC invasion and metastasis through upregulating FGFR4 and SRC. (A) Western blotting analysis of ELF4, FGFR4, SRC expression in the indicated CRC cells. (B) Transwell assays. (C-G) ELF4 promotes CRC lung metastasis through upregulating FGFR4 and SRC. (C) Representative bioluminescent images and dynamic intensity of bioluminescent signals. (D) Incidence of pulmonary metastases. (E) Number of lung metastatic nodules. (F) Overall survival. (G) H&E staining of lung tissues. (H-L) ELF4 facilitates CRC liver metastasis through transactivating FGFR4 and SRC. (H) Representative bioluminescent images and dynamic intensity of bioluminescent signals. (I) Incidence of liver metastases. (J) Number of tumor foci on liver surface. (K) Overall survival. (L) H&E staining of liver tissues. (M) A schematic diagram of the function of FGF19-ELF4-FGFR4 pathway in CRC metastasis.

Figure 2. ELF4 facilitates CRC invasion and metastasis through upregulating FGFR4 and SRC. (A) Western blotting analysis of ELF4, FGFR4, SRC expression in the indicated CRC cells. (B) Transwell assays. (C-G) ELF4 promotes CRC lung metastasis through upregulating FGFR4 and SRC. (C) Representative bioluminescent images and dynamic intensity of bioluminescent signals. (D) Incidence of pulmonary metastases. (E) Number of lung metastatic nodules. (F) Overall survival. (G) H&E staining of lung tissues. (H-L) ELF4 facilitates CRC liver metastasis through transactivating FGFR4 and SRC. (H) Representative bioluminescent images and dynamic intensity of bioluminescent signals. (I) Incidence of liver metastases. (J) Number of tumor foci on liver surface. (K) Overall survival. (L) H&E staining of liver tissues. (M) A schematic diagram of the function of FGF19-ELF4-FGFR4 pathway in CRC metastasis.

[Introduction]
Photodynamic therapy (PDT) is a treatment methodology in which tumor tissue incorporated with a photosensitizer (PS) is irradiated with incident light at an appropriate wavelength. This causes a photochemical reaction that releases free radicals, thus inducing tumor cell necrosis via oxidative stress. The second generation photosensitizer talaporfin is rapidly cleared from the skin and requires a shorter sun/shade period (2 weeks). Furthermore, the depth of the effect is expected to reach the deeper layers of the muscularis propria because of the diode laser excitation wavelength. PDT has great potential for curative treatment and therapy of early-stage cancer, but for advanced cancer, it remains a palliative therapy for local improvement; it is desirable to establish a combination therapy with surgery, radiation, and chemotherapy. It was reported that STING (Stimulator of interferon genes) regulates a transcriptional program that controls the generation of reactive oxygen species (ROS) and that STING loss alters ROS homeostasis to reduce DNA damage and cause therapeutic resistance. Thus, we predicted that pharmacological activation of STING would enhance DNA damage by PDT. Our study investigated the efficacy of talaporfin PDT combined with a STING agonist.
[Aims & methods]
1. We established STING knockout cells in a colon cancer cell line (HCT116) and incubated them with talaporfin followed by irradiation with 660 nm LED light. We then compared cell death-inducing and apoptosis-inducing effects between the parental and knockout cells.
2. We measured DNA damage by immunofluorescence microscopy, with an anti–g-H2AX antibody, after talaporfin PDT in the parental and knockout cells.
3. We investigated PDT-induced STING pathway activation of STING-regulated genes by Western blot analyses.
4. We assessed the in vivo efficacy of talaporfin PDT, STING agonist, and their combination in a xenograft tumor model.
[Results]
1. STING knockout cells significantly enhanced resistance to PDT, as measured by a cell survival assay. PDT in the parental cells caused significantly greater apoptosis than in STING knockout cells.
2. Treating parental cell lines with talaporfin PDT led to significantly more γ-H2AX foci formation than in STING knockout cells, as measured via fluorescence microscopy.
3. At the protein level, STING dependent signaling was enhanced by PDT, with enhanced TBK-1 phosphorylation, and the effects that were eliminated by STING loss.
4. The combination exhibited significantly greater tumor growth inhibition than by single agents alone in a xenograft tumor model.
[Conclusion]
Our results reveal that a STING agonist enhances the efficacy of talaporfin PDT by regulating the amount of DNA damage. We believe that the combination of talaporfin PDT with a STING agonist provides a potential effective candidate for future antitumor therapies.

Takaya Shimura

Appearances