Regenerative proliferation of differentiated cells by mTORC1-dependent paligenosis.

In 1900, Adami speculated that a sequence of context-independent energetic and structural changes governed the reversion of differentiated cells to a proliferative, regenerative state. Accordingly, we show here that differentiated cells in diverse organs become proliferative via a shared program. Metaplasia-inducing injury caused both gastric chief and pancreatic acinar cells to decrease mTORC1 activity and massively upregulate lysosomes/autophagosomes; then increase damage associated metaplastic genes such as Sox9; and finally reactivate mTORC1 and re-enter the cell cycle. Blocking mTORC1 permitted autophagy and metaplastic gene induction but blocked cell cycle re-entry at S-phase. In kidney and liver regeneration and in human gastric metaplasia, mTORC1 also correlated with proliferation. In lysosome-defective Gnptab-/- mice, both metaplasia-associated gene expression changes and mTORC1-mediated proliferation were deficient in pancreas and stomach. Our findings indicate differentiated cells become proliferative using a sequential program with intervening checkpoints: (i) differentiated cell structure degradation; (ii) metaplasia- or progenitor-associated gene induction; (iii) cell cycle re-entry. We propose this program, which we term "paligenosis", is a fundamental process, like apoptosis, available to differentiated cells to fuel regeneration following injury.

DDIT4 Licenses Only Healthy Cells to Proliferate During Injury-induced Metaplasia.

BACKGROUND AND AIMS: In stomach, metaplasia can arise from differentiated chief cells that become mitotic via paligenosis, a stepwise program. In paligenosis, mitosis initiation requires reactivation of the cellular energy hub mTORC1 after initial mTORC1 suppression by DNA damage induced transcript 4 (DDIT4 aka REDD1). Here, we use DDIT4-deficient mice and human cells to study how metaplasia increases tumorigenesis risk. METHODS: A tissue microarray of human gastric tissue specimens was analyzed by immunohistochemistry for DDIT4. C57BL/6 mice were administered combinations of intraperitoneal injections of high-dose tamoxifen (TAM) to induce spasmolytic polypeptide-expressing metaplasia (SPEM) and rapamycin to block mTORC1 activity, and N-methyl-N-nitrosourea (MNU) in drinking water to induce spontaneous gastric tumors. Stomachs were analyzed for proliferation, DNA damage, and tumor formation. CRISPR/Cas9-generated DDIT4-/- and control human gastric cells were analyzed for growth in vitro and in xenografts with and without 5-fluorouracil (5-FU) treatment. RESULTS: DDIT4 was expressed in normal gastric chief cells in mice and humans and decreased as chief cells became metaplastic. Paligenotic Ddit4-/- chief cells maintained constitutively high mTORC1, causing increased mitosis of metaplastic cells despite DNA damage. Lower DDIT4 expression correlated with longer survival of patients with gastric cancer. 5-FU-treated DDIT4-/- human gastric epithelial cells had significantly increased cells entering mitosis despite DNA damage and increased proliferation in vitro and in xenografts. MNU-treated Ddit4-/- mice had increased spontaneous tumorigenesis after multiple rounds of paligenosis induced by TAM. CONCLUSIONS: During injury-induced metaplastic proliferation, failure of licensing mTORC1 reactivation correlates with increased proliferation of cells harboring DNA damage, as well as increased tumor formation and growth in mice and humans.

Racial and ethnic disparities in 21-gene recurrence scores, chemotherapy, and survival among women with hormone receptor-positive, node-negative breast cancer.

PURPOSE: Cutoffs of the 21-gene recurrence score (RS), a commonly used genomic assay for hormone receptor-positive breast cancer, have been updated. Little is known about racial/ethnic differences in RS results, RS-guided chemotherapy use, and outcomes on updated cutoff (RS ≥ 31 defined as high-risk) in the real-world setting. METHODS: A total of 81,937 women [75.0% whites, 7.7% blacks, 8.3% Asian American/Pacific Islanders (AAPIs), and 9.0% Hispanics] diagnosed with hormone receptor-positive breast cancer between 2004 and 2015, who received the 21-gene assay, were identified from the Surveillance, Epidemiology, and End Results. Logistic regressions estimated the race-associated odds ratios (ORs) of RS and chemotherapy use. Cox regressions estimated the race-associated hazard ratios (HRs) of breast cancer-specific and all-cause mortality. RESULTS: Compared with white women, black women were more likely to have RS-defined high-risk tumors (adjusted OR [aOR] 1.29; 95% CI 1.16-1.42). In high RS, blacks had lower odds of chemotherapy use (aOR 0.76; 95% CI 0.62-0.94) than whites, particularly among women ≥ 65 years (aOR 0.51; 95% CI 0.35-0.76), while AAPI and Hispanic women had no variation in chemotherapy use compared with whites in high RS. Black women had a higher risk of breast cancer-specific mortality (HR 1.37; 95% CI 1.12-1.67) and all-cause mortality compared with white women after adjusting for demographic and pathological factors, county-level socioeconomic deprivation, treatments and RS; AAPIs had lower mortality and Hispanics had similar mortality. CONCLUSIONS: Black women were more likely to have a high-risk RS tumor and less likely to receive chemotherapy in the group of high RS, especially those ≥ 65 years. Further studies are needed to identify barriers to chemotherapy in black patients with high RS scores.

miR-148a inhibits early relapsed colorectal cancers and the secretion of VEGF by indirectly targeting HIF-1α under non-hypoxia/hypoxia conditions.

Vascular endothelial growth factor (VEGF) is correlated with angiogenesis and early relapse of colorectal cancer (CRC). This study investigated the role of miR-148a in the regulation of VEGF/angiogenesis and early relapse of CRC. We established a stable clone with miR-148a expression in HCT116 and HT29 cell lines and created a hypoxic condition by using CoCl2 to determine the underlying mechanism of miR-148a. The effects of miR-148a on the phosphoryl-ERK (pERK)/hypoxia-inducible factor-1α (HIF-1α)/VEGF pathway were evaluated through Western blotting and the inhibitory effect of miR-148a on angiogenesis was demonstrated through a tube formation assay. Sixty-three CRC tissues (28 early relapse and 35 non-early relapse) were analysed to assess the relationship between miR-148a and HIF-1α/VEGF. The protein expression of pERK/HIF-1α/VEGF in HCT116 and HT29 cells was significantly decreased by miR-148a (all P < 0.05). The protein expression of VEGF/HIF-1α was strongly inversely associated with the expression of miR-148a in the 63 CRC tissue samples (all P < 0.05). Tube formation assay demonstrated that miR-148a significantly obliterated angiogenesis. miR-148a suppresses VEGF through down-regulation of the pERK/HIF-1α/VEGF pathway and might lead to the inhibition of angiogenesis; miR-148a down-regulation increased the early relapse rate of CRC. This demonstrates that miR-148a is a potential diagnostic and therapeutic target.

Smoking status and subsequent gastric cancer risk in men compared with women: a meta-analysis of prospective observational studies.

BACKGROUND: Smoking is one of the well-established risk factors for gastric cancer incidence, yet whether men are more or equally susceptible to gastric cancer due to smoking compared with women is a matter of controversy. The aim of this study was to investigate and compare the effect of sex on gastric cancer risk associated with smoking. METHODS: We conducted a systemic literature search in MEDLINE, EMBASE, and the Cochrane CENTRAL databases to identify studies published from inception to December 2018. We included prospective observational studies which reported effect estimates with 95% confidence intervals (CIs) for associations of current or former smokers with the incidence of gastric cancer by sex. We calculated the ratio of relative risk (RRR) with corresponding 95% CI based on sex-specific effect estimates for current or former smokers versus non-smokers on the risk of gastric cancer. RESULTS: We included 10 prospective studies with 3,381,345 participants in our analysis. Overall, the summary RRR (male to female) for gastric cancer risk in current smokers was significantly increased compared with non-smokers (RRR: 1.30; 95% CI: 1.05-1.63; P = 0.019). Furthermore, there was no significant sex difference for the association between former smokers and gastric cancer risk (RRR: 1.20; 95% CI: 0.92-1.55; P = 0.178). However, the result of sensitivity analysis indicated the pooled result was not stable, which was altered by excluding a nested case-control study (RRR: 1.31; 95% CI: 1.10-1.57; P = 0.002). CONCLUSION: This systematic review showed a potential sex difference association between current smokers and the risk of gastric cancer. The sex differential in smokers can give important clues for the etiology of gastric cancers and should be examined in further studies.

The role of EGFR mutation as a prognostic factor in survival after diagnosis of brain metastasis in non-small cell lung cancer: a systematic review and meta-analysis.

BACKGROUND: The brain is a common site for metastasis in non-small-cell lung cancer (NSCLC). This study was designed to evaluate the relationship between the mutational of the epidermal growth factor receptor (EGFR) and overall survival (OS) in NSCLC patients with brain metastases. METHODS: Searches were performed in PubMed, EmBase, and the Cochrane Library to identify studies evaluating the association of EGFR mutation with OS in NSCLC patients through September 2017. RESULTS: 4373 NSCLC patients with brain metastases in 18 studies were involved. Mutated EGFR associated with significantly improved OS compared with wild type. Subgroup analyses suggested that this relationship persisted in studies conducted in Eastern, with retrospective design, with sample size ≥500, mean age of patients ≥65.0 years, percentage male < 50.0%, percentage of patients receiving tyrosine kinase inhibitor ≥30.0%. Finally, although significant publication bias was observed using the Egger test, the results were not changed after adjustment using the trim and fill method. CONCLUSIONS: This meta-analysis suggests that EGFR mutation is an important predictive factor linked to improved OS for NSCLC patients with brain metastases. It can serve as a useful index in the prognostic assessment of NSCLC patients with brain metastases.

Downregulation of RASSF6 promotes breast cancer growth and chemoresistance through regulation of Hippo signaling.

This study aims to investigate the clinical significance and biological function of RASSF6 in human breast cancers. RASSF6 protein was found to be downregulated in 42 of 95 human breast cancer tissues by immunohistochemistry, which was associated with advanced TNM stage and nodal metastasis. The rate of RASSF6 downregulation was higher in Triple-negative breast cancer (TNBC). Downregulation of RASSF6 protein was also found in breast cancer cell lines, especially in TNBC cell lines. Overexpression RASSF6 inhibited cell growth rate and colony formation ability in MDA-MB-231 cell line. Depletion of RASSF6 promoted proliferation rate and colony formation ability in T47D cell line. Flow cytometry/PI staining demonstrated that RASSF6 inhibited cell cycle transition. AnnxinV/PI analysis showed that RASSF6 overexpression upregulated apoptosis induced by cisplatin (CDDP) while RASSF6 depletion inhibited apoptosis. JC-1 staining showed that RASSF6 overexpression inhibited mitochondrial membrane potential. Western blot analysis demonstrated that RASSF6 repressed cyclin D1, YAP while upregulated p21, cleaved caspase 3 and cytochrome c expression. In addition, RASSF6 activated Hippo signaling pathway by upregulating MST1/2 and LATS1 phosphorylation. Restoration of YAP inhibited cleaved caspase 3 and cytochrome c which were induced by RASSF6. Restoration of YAP also reduced the rate of CDDP induced apoptosis. In conclusion, this study provided evidence that RASSF6 functions as a potential tumor suppressor in human breast cancer through activation of Hippo pathway.

Effect of neoadjuvant chemotherapy in patients with gastric cancer: a PRISMA-compliant systematic review and meta-analysis.

BACKGROUND: Neoadjuvant chemotherapy (NAC) is extensively used in the treatment of patients with gastric cancer (GC), particularly in high risk, advanced gastric cancer. Previous trials testing the efficacy of NAC have reported inconsistent results. METHODS: This study compares the combined use of NAC and surgery with surgery alone for GC by using a meta-analytic approach. We performed an electronic search of PubMed, EmBase, and the Cochrane Library to identify randomized controlled trials (RCTs) on NAC published before Oct 2015. The primary outcome of the studies was data on survival rates for patients with GC. The summary results were pooled using the random-effects model. We included 12 prospective RCTs reporting data on 1538 GC patients. RESULTS: Patients who received NAC were associated with significant improvement of OS (P = 0.001) and PFS (P < 0.001). Furthermore, NAC therapy significantly increased the incidence of 1-year survival rate (SR) (P = 0.020), 3-year SR (P = 0.011), and 4-year SR (P = 0.001). Similarly, NAC therapy was associated with a lower incidence of 1-year (P < 0.001), 2-year (P < 0.001), 3-year (P < 0.001), 4-year (P = 0.001), and 5-year recurrence rate (P = 0.002). Conversely, patients who received NAC also experienced a significantly increased risk of lymphocytopenia (P = 0.003), and hemoglobinopathy (P = 0.021). CONCLUSIONS: The findings of this study suggested that NAC is associated with significant improvement in the outcomes of survival and disease progression for GC patients while also increasing some toxicity.

The efficacy and safety of targeted therapy with or without chemotherapy in advanced gastric cancer treatment: a network meta-analysis of well-designed randomized controlled trials.

BACKGROUND: Advanced gastric cancer (AGC) is a severe malignant tumor associated with high mortality. Targeted therapy is an important approach for improving the therapeutic effects of AGC treatment. This study evaluates the efficacy and safety of targeted agents for AGC patients. METHODS: PubMed, EmBase, and the Cochrane Library were searched for double-blind randomized controlled trials (RCTs) of AGC treatments published prior to July 2017. Progression-free survival (PFS), overall survival (OS), objective response rate (ORR), and severe adverse effects (AEs) were evaluated to determine the efficacy and safety of targeted agents. A network meta-analysis with a frequentist framework was performed to assess the effects of various targeted agents for AGC treatment. RESULTS: Our analysis included 16 articles involving 5371 patients and 11 types of agents. The network meta-analysis showed that apatinib (97.5%) was most likely to improve PFS, followed by regorafenib (86.3%) and rilotumumab (65.4%). Apatinib was similarly best for OS outcome, (95.5%) followed by rilotumumab (74.7%) and regorafenib (70%). Apatinib (89.6%) also had the best improvement on ORR, followed by rilotumumab (75.4%) and everolimus (68.4%). Bevacizumab (85.5%) was likely to get the lowest severe AEs, followed by sunitinib (63%). CONCLUSIONS: Apatinib, regorafenib, and rilotumumab improved patient PFS and OS. When combined with chemotherapy, ramucirumab and rilotumumab had high efficacy but low tolerability, and bevacizumab had moderate efficacy and tolerability for PFS. Without chemotherapy, ramucirumab and regorafenib had relatively high therapeutic efficacy tolerability for PFS.

Increased IFRD1 Expression in Human Colon Cancers Predicts Reduced Patient Survival.

BACKGROUND: Colon cancer (CRC) is the third most common cancer worldwide. CRC develops through combinations of genetic and epigenetic changes. However, there is marked heterogeneity in the "driver gene" mutational profiles within and among colon cancers from individual patients, and these are not sufficient to explain differences in colon cancer behavior and treatment response. Global modulation of the tumor landscape may play a role in cancer behavior. Interferon-related developmental regulator 1 (IFRD1) is a transcriptional co-regulator that modulates expression of large gene cassettes and plays a role in gut epithelial proliferation following massive intestinal resection. AIMS: We address the hypothesis that increased IFRD1 expression in colon cancers is associated with poorer patient survival. METHODS: Tumor and normal tissue from colon cancer patient cohorts from the USA, Spain, and China were used for this study. Cancers were scored for the intensity of IFRD1 immunostaining. The primary clinical outcome was overall survival defined as time from diagnosis to death due to cancer. Kaplan-Meier method and log-rank analysis were used to assess the association between IFRD1 expression and survival. RESULTS: Almost all (98.7%) colon cancers showed readily detectable IFRD1 expression, with immunoreactivity primarily in the tumor cytoplasm. High IFRD1 colon cancer expression was significantly associated with decreased 5-year patient survival. Patients in the American cohort with high IFRD1 expression had a poorer prognosis. CONCLUSIONS: We have demonstrated that high IFRD1 protein expression in colon cancer is associated with poorer patient prognosis, suggesting a potential role for IFRD1 in modulating tumor behavior.

Development of a deregulating microRNA panel for the detection of early relapse in postoperative colorectal cancer patients.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer mortality worldwide and is associated with high recurrence and mortality, despite recent advancements in therapeutic strategies. MicroRNA (miR) deregulation is associated with CRC development and recurrence; therefore, miRs may be reliable biomarkers for detecting early relapse postoperatively. METHODS: In this study ten candidates were identified using miR arrays: miR-7, miR-31, miR-93, miR-141, miR-195, miR-375, miR-429, miR-494, miR-650, and let-7b. Substantial differences were observed in their expression levels between early relapsed (recurrences within 12 months after surgery) and non-early relapsed CRC patients. The validation study, including 50 early relapsed and 54 non-early relapsed patients, confirmed miR expression alterations in cancer tissue samples. RESULTS: Using a miR real-time quantitative polymerase chain reaction (RT-qPCR), we observed that expression levels of miR-93, miR-195, and let-7b were significantly decreased, whereas those of miR-7, miR-141 and miR-494 showed increases that were more significant in the CRC tissue samples from the early relapsed patients than in those from the non-early relapsed patients. Disease-free survival and overall survival were significantly worse in the high miR-7, miR-141, and miR-494 expression subgroups and the low miR-93 and miR-195 expression subgroups (all P < 0.05). A panel of 6 miRs (miR-7, miR-93, miR-195, miR-141, miR-494, and let-7b), at a cut-off value of 2 deregulated miRs, distinguished early relapsed CRC from non-early relapsed CRC, with a sensitivity of 76.6 % and a specificity of 71.4 %. By combining this 6-miRs panel with 6 clinicopathologic factors, at a cut-off value of 4, distinguished early relapsed CRC from non-early relapsed CRC, with a sensitivity of 89.4 % and a specificity of 88.9 %. CONCLUSIONS: This study showed that the developed miR panel has the potential to improve predicting early relapse in CRC patients.

Borrmann type IV gastric cancer should be classified as pT4b disease.

BACKGROUND: The impact of macroscopic pathologic features of primary tumor that could be obtained preoperatively on pT classification has not been reported so far. The aim of this study was to investigate the feasibility of incorporation of Borrmann type IV gastric cancer into the pT classification. MATERIALS AND METHODS: Clinicopathologic and prognostic data of 1622 patients with advanced gastric cancer who underwent radical surgery were retrospectively studied. RESULTS: Of 1622 patients, 135 (8.32%) patients were classified as having Borrmann type IV gastric cancer. We first confirmed that Borrmann type IV gastric cancer was one of the independent prognostic factors for patients with advanced gastric cancer who underwent radical surgery. Interestingly, we found that overall survival of patients with Borrmann type IV gastric cancer could be clearly distinguished by pN classification and pathological TNM stage but not by pT classification. Importantly, further analysis demonstrated that the prognosis of Borrmann type IV gastric cancers was homogeneous with that of pT4b cancers but poorer than pT2, pT3, pT4a cancers. Therefore, we proposed a novel pT classification in which pT4b disease was defined as cancers that were Borrmann type IV or those that had invaded adjacent structures. Two-step multivariate analysis demonstrated that the novel pT classification was more suitable for prognostic assessment than the original classification. CONCLUSIONS: Classifying Borrmann type IV gastric cancer as pT4b disease improves pT classification prediction of prognosis in patients with advanced gastric cancer after radical surgery.

Lung cancer cells induce senescence and apoptosis of pleural mesothelial cells via transforming growth factor-beta1.

Pleural dissemination is commonly associated with metastatic advanced lung cancer. The injury of pleural mesothelial cells (PMCs) by soluble factors, such as transforming growth factor-beta1 (TGF-ß1), is a major driver of lung cancer pleural dissemination (LCPD). In this study, we examine the effects of TGF-ß1 on PMC injury and the ability of TGF-ß1 inhibition to alleviate this effect both in vitro and in vivo. PMCs were co-cultured with the high TGF-ß1-expressing lung cancer cell line A549 and with various TGF-ß1 signaling inhibitors. Expression of cleaved-caspase 3, cleaved-caspase 9, p21, and p16 were evaluated by Western blot and immunofluorescent confocal imaging. Apoptosis was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltrazoliumbromide assay and AnnexinV-propidium iodide (PI) staining. PMC senescence was assessed by staining for senescence-associated ß-galactosidase (SA-ß-Gal). The ability of lung cancer cells (LCCs) to adhere to injured PMCs was investigated using an LCC-PMC adhesion assay. In our mouse model, PMC injury status was monitored by hematoxylin-eosin (H&E) and Masson's trichrome staining. LCCs expressing high levels of TGF-ß1 induce apoptosis and senescence of PMCs in a co-culture system. Injured PMCs adhere to LCCs, which may further promote LCPD. Importantly, PMC monolayer injury could be reversed with TGF-ß1 inhibitors. This was consistent with our in vivo data showing that the TGF-ß1 inhibitor SB-431542 attenuated PMC barrier injury induced by A549 culture medium in our mouse model. Our study highlights the importance of TGF-ß1 signaling in LCPD and establishes this signaling pathway as a potential therapeutic target in the disease.

Peritoneal milky spots serve as a hypoxic niche and favor gastric cancer stem/progenitor cell peritoneal dissemination through hypoxia-inducible factor 1α.

Peritoneal dissemination is the most common cause of death in gastric cancer patients. The hypoxic microenvironment plays a major role in controlling the tumor stem cell phenotype and is associated with patients' prognosis through hypoxia-inducible factor-1α (HIF-1α), a key transcriptional factor that responds to hypoxic stimuli. During the peritoneal dissemination process, gastric cancer stem/progenitor cells (GCSPCs) are thought to enter into and maintained in peritoneal milky spots (PMSs), which have hypoxic microenvironments. However, the mechanism through which the hypoxic environment of PMSs regulated GCSPC maintenance is still poorly understood. Here, we investigated whether hypoxic PMSs were an ideal cancer stem cell niche suitable for GCSPC engraftment. We also evaluated the mechanisms through which the HIF-1α-mediated hypoxic microenvironment regulated GCSPC fate. We observed a positive correlation between HIF-1α expression and gastric cancer peritoneal dissemination (GCPD) in gastric cancer patients. Furthermore, the GCSPC population expanded in primary gastric cancer cells under hypoxic condition in vitro, and hypoxic GCSPCs showed enhanced self-renewal ability, but reduced differentiation capacity, mediated by HIF-1α. In an animal model, GCSPCs preferentially resided in the hypoxic zone of PMSs; moreover, when the hypoxic microenvironment in PMSs was destroyed, GCPD was significantly alleviated. In conclusion, our results demonstrated that PMSs served as a hypoxic niche and favored GCSPCs peritoneal dissemination through HIF-1α both in vitro and in vivo. These results provided new insights into the GCPD process and may lead to advancements in the clinical treatment of gastric cancer.

Tumor-associated mesothelial cells are negative prognostic factors in gastric cancer and promote peritoneal dissemination of adherent gastric cancer cells by chemotaxis.

Peritoneal dissemination is highly frequent in gastric cancer. Damage to human peritoneal mesothelial cell (HPMC) barriers provokes gastric cancer peritoneal dissemination (GCPD), the key events during GCPD, is characterized by fibroblastic development. In this study, we have studied the association between fibroblast activation protein (FAP) expression in peritoneum and the pathological features of the primary tumor. The clinical prognosis of gastric cancer patients was evaluated according to FAP expression. In a gastric cancer cell-HPMC co-culture system, expression of E-cadherin, α-smooth muscle actin, and FAP were evaluated by Western blotting. Gastric cancer cell migration and adhesion to HPMC were also assayed. Our results showed positive peritoneal staining of FAP in 36/86 cases (41.9 %), which was associated with a higher TNM stage in primary gastric cancer and higher incidence of GCPD (both p<0.05). Survival analysis showed FAP expression was an independent prognostic factor of poor survival (p=0.02). Peritoneum of FAP-positive expression exhibited a distinct fibrotic development and expressed higher level of the mesenchymal marker α-SMA, which was confirmed by the in vitro Western blot assay. In HPMC and gastric cancer cell adherence assay, SGC-7901 cells preferentially adhered to TA-HPMC at different cell densities (both p<0.05). Additionally, SGC-7901 cells were more prone to chemotaxis by FAP-expressed tumor-associated-human peritoneal mesothelial cells (TA-HPMC) compared with HPMC co-cultured with normal gastric glandular epithelial cells in a time-dependent manner (both p<0.05). Our study indicated a positive correlation between peritoneum FAP expression and GCPD. FAP-expressed TA-HPMC might be an important cellular component and instigator of GCPD.

Transforming growth factor-beta1 signaling blockade attenuates gastric cancer cell-induced peritoneal mesothelial cell fibrosis and alleviates peritoneal dissemination both in vitro and in vivo.

Peritoneal dissemination is the most frequent metastatic pattern of advanced gastric cancer and the main cause of death in gastric cancer patients. Transforming growth factor-beta1 (TGF- ß1), one of the most potent fibrotic stimuli for human peritoneal mesothelial cells, has been shown to play an important role in this process. In this study, we investigated the effect of TGF- ß1 signaling blockade in gastric cancer cell (GCC)-induced human peritoneal mesothelial cell (HPMC) fibrosis. HPMCs were cocultured with the high TGF- ß1 expressing GCC line SGC-7901 and various TGF- ß1 signaling inhibitors or SGC-7901 transfected with TGF-ß1-specific siRNA. HPMC fibrosis was monitored on the basis of morphology. Expression of the epithelial cell marker, E-cadherin, and the mesenchymal marker, α-smooth muscle actin (α-SMA), was evaluated by Western blotting and immunofluorescence confocal imaging. GCC adhesion to HPMC was also assayed. In nude mouse tumor model, the peritoneal fibrotic status was monitored by immunofluorescent confocal imaging and Masson's trichrome staining; formation of metastatic nodular and ascites fluid was also evaluated. Our study demonstrated that GCC expressing high levels of TGF-ß1 induced HMPC fibrosis, which is characterized by both upregulation of E-cadherin and downregulation of α-SMA. Furthermore, HPMC monolayers fibrosis was reversed by TGF- ß1 signaling blockade. In vivo, the TGF- ß1 receptor inhibitor SB-431542 partially attenuated early-stage gastric cancer peritoneal dissemination (GCPD). In conclusion, our study confirms the significance of TGFß1 signaling blockade in attenuating GCPD and may provide a therapeutic target for clinical therapy.

The mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa shift peritoneal milky spot macrophages towards an M1 phenotype to dampen peritoneal dissemination.

Peritoneal dissemination (PD) of tumor cells is the most frequent pattern of gastric cancer recurrence and the leading cause of death. Peritoneal milky spots are deemed as the site of origin of gastric cancer PD wherein the main cellular components are macrophages. A vaccine derived from the mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA) has exhibit strong immune modulatory properties. In the present study, we tested the hypothesis whether the PA-MSHA vaccine activated peritoneal milky spot macrophages (PMSM) in a manner that would attenuate PD. It was observed that PA-MSHA activated PMSM towards a classical activation phenotype via a toll-like receptor4/9-dependent mechanism, which increased interleukin-12 levels and promoted the expression of co-stimulatory and antigen-presenting molecules like CD80, CD86, and MHC-II (P < 0.05). In addition, PA-MSHA-treated PMSM exhibited strong nonspecific antitumor effects in both contact-dependent and contact-independent modes of action (P < 0.05). In mice treated with PA-MSHA before inoculating gastric cancer cells, we noted alleviated PD toward the untreated mice. In conclusion, our findings demonstrated that PA-MSHA can stimulate PMSM towards an M1 phenotype and that activated PMSM inhibit gastric cancer growth and PD both in vitro and in vivo. The results of the current study provide a mechanistic insight that is relevant to the potential application of PA-MSHA in the treatment of gastric cancer-mediated PD.

Influence of different hypoxia models on metastatic potential of SGC-7901 gastric cancer cells.

Gastric cancers are one of the most common gastrointestinal tumors with proclivity to metastasis. Hypoxia is an important feature in solid tumor microenvironment; accumulating evidence suggests that chronic and repetitive exposure to hypoxia and reoxygenation seems to provide an advantage to tumor growth. However, mechanisms of intermittent hypoxia regulating gastric cancer metastatic potential remain to be elucidated. In the present study, we established the continuous and intermittent gastric cancer hypoxia models, and we compared the effects of these models on gastric cancer cell metastatic potential. Hypoxia-inducible factor-1α (HIF-1α) and hypoxia target protein/genes expression in response to different hypoxia models were analyzed by Western blot, immunofluorescence, and real-time PCR assays. Gastric cancer cell migration and invasion ability were analyzed by wound healing and Boyden chamber assay. Colony formation and tumor spheroid formation assays were used to assess the ability of self-renewal. Stem-related protein OCT4 and HIF-1α expression were also analyzed by confocal immunofluorescence in single tertiary generation spheroid. We demonstrated that the intermittent hypoxia model upregulated expression of both HIF-1α and hypoxia target protein/genes in a time-dependent manner compared with that of SGC-7901 cells cultured under normoxia. Remarkably, HIF-1α protein was more prone to being located in the nucleus of SGC-7901 cells under conditions of intermittent hypoxia. Gastric cancer cells' migration and invasion ability were significantly enhanced after hypoxic culture; moreover, intermittent hypoxia-conditioned SGC-7901 cells exhibited higher invasiveness than the continuous hypoxia-conditioned SGC-7901 cells. Gastric cancer stem/progenitor cell subpopulation was also enlarged which indicated an enhanced self-renewal ability in hypoxic cultured SGC-7901 cells. Our study emphasizes the significance of hypoxia in regulating gastric cancer cell metastasis potential. Compared with continuous hypoxia, intermittent hypoxia is a more effective and potent hypoxic stimulus. These results provide a new insight to investigate the hypoxic microenvironment within solid tumors.

Gastric intestinal metaplasia: progress and remaining challenges.

Most gastric cancers arise in the setting of chronic inflammation which alters gland organization, such that acid-pumping parietal cells are lost, and remaining cells undergo metaplastic change in differentiation patterns. From a basic science perspective, recent progress has been made in understanding how atrophy and initial pyloric metaplasia occur. However, pathologists and cancer biologists have long been focused on the development of intestinal metaplasia patterns in this setting. Arguably, much less progress has been made in understanding the mechanisms that lead to the intestinalization seen in chronic atrophic gastritis and pyloric metaplasia. One plausible explanation for this disparity lies in the notable absence of reliable and reproducible small animal models within the field, which would facilitate the investigation of the mechanisms underlying the development of gastric intestinal metaplasia (GIM). This review offers an in-depth exploration of the current state of research in GIM, shedding light on its pivotal role in tumorigenesis. We delve into the histological subtypes of GIM and explore their respective associations with tumor formation. We present the current repertoire of biomarkers utilized to delineate the origins and progression of GIM and provide a comprehensive survey of the available, albeit limited, mouse lines employed for modeling GIM and engage in a discussion regarding potential cell lineages that serve as the origins of GIM. Finally, we expound upon the myriad signaling pathways recognized for their activity in GIM and posit on their potential overlap and interactions that contribute to the ultimate manifestation of the disease phenotype. Through our exhaustive review of the progression from gastric disease to GIM, we aim to establish the groundwork for future research endeavors dedicated to elucidating the etiology of GIM and developing strategies for its prevention and treatment, considering its potential precancerous nature.

Metaplastic regeneration in the mouse stomach requires a reactive oxygen species pathway.

In pyloric metaplasia, mature gastric chief cells reprogram via an evolutionarily conserved process termed paligenosis to re-enter the cell cycle and become spasmolytic polypeptide-expressing metaplasia (SPEM) cells. Here, we use single-cell RNA sequencing (scRNA-seq) following injury to the murine stomach to analyze mechanisms governing paligenosis at high resolution. Injury causes induced reactive oxygen species (ROS) with coordinated changes in mitochondrial activity and cellular metabolism, requiring the transcriptional mitochondrial regulator Ppargc1a (Pgc1α) and ROS regulator Nf2el2 (Nrf2). Loss of the ROS and mitochondrial control in Ppargc1a-/- mice causes the death of paligenotic cells through ferroptosis. Blocking the cystine transporter SLC7A11(xCT), which is critical in lipid radical detoxification through glutathione peroxidase 4 (GPX4), also increases ferroptosis. Finally, we show that PGC1α-mediated ROS and mitochondrial changes also underlie the paligenosis of pancreatic acinar cells. Altogether, the results detail how metabolic and mitochondrial changes are necessary for injury response, regeneration, and metaplasia in the stomach.