Abstract Background Colorectal cancer recurrence is largely attributed to dormant tumor cells that evade therapy. The role of organelle metabolism, particularly lipid droplets (LDs) accumulation, in maintaining tumor dormancy remains poorly understood. This study aimed to investigate how LDs contribute to dormancy and to identify key regulatory mechanisms. Methods Using patient-derived xenograft models and colorectal cancer cell lines, we established chemotherapy-induced dormant, proliferative, and recurrent cells. Transcriptomic profiling identified key regulatory genes and lipidomics of LDs was performed by Liquid Chromatography‑Tandem Mass Spectrometry. Functional validation was performed via genetic knockdown/overexpression, pharmacological inhibition, and mutational analysis. Lipid droplet content, reactive oxygen species levels, and dormancy markers were assessed using staining, flow cytometry, and immunoblotting. Clinical correlation was evaluated using patient datasets and survival analysis. Results Dormant cells exhibited elevated LD levels compared to proliferative or recurrent cells. Transcriptomic analysis identified ACSL5 as a key upregulated gene in dormant cells. ACSL5 promoted LD accumulation, which in turn reduced intracellular reactive oxygen species and stabilized dormancy by sequestering oxidized lipids. The transcription factor ELF1 directly bound the ACSL5 promoter, establishing the ELF1-ACSL5 regulatory axis. Disruption of this axis depleted LDs, increased ROS, and forced dormant cells to exit dormancy. Clinically, high ACSL5 expression correlated with poor prognosis in colorectal cancer patients. Conclusion This study defines a novel ELF1-ACSL5-lipid droplet axis that maintains colorectal cancer dormancy by promoting lipid sequestration and redox homeostasis. These findings reveal organelle-centric metabolic reprogramming as a fundamental dormancy mechanism and nominate this axis as a promising therapeutic target to prevent cancer recurrence.

Chemotherapy is a crucial treatment for colorectal tumors. However, its efficacy is restricted by chemoresistance. Recently, Golgi dispersal has been suggested to be a potential response to chemotherapy, particularly to drugs that induce DNA damage. However, the underlying mechanisms by which Golgi dispersal enhances the capacity to resist DNA-damaging agents remain unclear. Here, we demonstrated that DNA-damaging agents triggered Golgi dispersal in colorectal cancer (CRC), and cancer stem cells (CSCs) possessed a greater degree of Golgi dispersal compared with differentiated cancer cells (non-CSCs). We further revealed that Golgi dispersal conferred resistance against the lethal effects of DNA-damaging agents. Momentously, Golgi dispersal activated the Golgi stress response via the PKCα/GSK3α/TFE3 axis, resulting in enhanced protein and vesicle trafficking, which facilitated drug efflux through ABCG2. Identification of Golgi dispersal indicated an unexpected pathway regulating chemoresistance in CRC.

Tumor deposits (TDs) represent a specific form tumor metastasis observed in colorectal cancer (CRC). The lack of successfully established cell lines for TDs, as well as the molecular mechanisms by which TDs occur remain largely unknown. Here, we established paired CRC organoids, including a human primary cancer organoid and its TD organoid, from a 46-year-old male patient with CRC. Further analysis revealed that, compared with primary tumor-derived cells, TD-derived cells exhibited enhanced proliferative, invasive and metastatic capabilities, and increased expression of stemness-related proteins. Furthermore, the present findings also demonstrated that TD-derived cells were more resistant to oxaliplatin or 5-FU. Transcriptomic profiling and qPCR revealed that TD-derived cells exhibited more alterations in fatty acid metabolism signaling and enhanced lipid synthesis ability compared to primary tumor-derived cells. Inhibition of lipid synthesis markedly decreased resistance to oxaliplatin in TD-derived cells. Taken together, the paired organoids established using CRC primary tumor and its TD specimens will provide valuable tools to study tumorigenicity, metastasis and chemoresistance in CRC. Notably, these models will provide novel insights to study tumor heterogeneity and lipid metabolism in CRC.

Abnormal nuclear enlargement is a diagnostic and physical hallmark of malignant tumors. Large nuclei are positively associated with an increased risk of developing metastasis; however, a large nucleus is inevitably more resistant to cell migration due to its size. The present study demonstrated that the nuclear size of primary colorectal cancer (CRC) cells at an advanced stage was larger than cells at an early stage. In addition, the nuclei of CRC liver metastases were larger than those of the corresponding primary CRC tissues. CRC cells were sorted into large-nucleated cells (LNCs) and small-nucleated cells (SNCs). Purified LNCs exhibited greater constricted migratory and metastatic capacity than SNCs in vitro and in vivo. Mechanistically, ErbB4 was highly expressed in LNCs, which phosphorylated lamin A/C at serine 22 via the ErbB4-Akt1 signaling pathway. Furthermore, the level of phosphorylated lamin A/C was a negative determinant of nuclear stiffness. Taken together, CRC LNCs possessed greater constricted migratory and metastatic potential than SNCs due to ErbB4-Akt1-mediated lamin A/C phosphorylation and nuclear softening. These results may provide a potential treatment strategy for tumor metastasis by targeting nuclear stiffness in patients with cancer, particularly CRC.

Invadopodia, being actin-rich membrane protrusions, play a vital role in tumor cell invasion and metastasis. Our previous studies have revealed some functions of the DOC-2/DAB2 interacting protein (DAB2IP) as a tumor suppressor. Nevertheless, the specific role and mechanism of DAB2IP in invadopodia formation remain unclear. Here, we find that DAB2IP effectively suppresses invadopodia formation and metastasis in breast cancer, both in vitro and in vivo. Additionally, DAB2IP could downregulate anaplastic lymphoma kinase (ALK), resulting in the inhibition of tyrosine phosphorylation of Cortactin and the prevention of invadopodia formation. DAB2IP competitively antagonizes the interaction between the deubiquitinating enzyme Ubiquitin-specific peptidase 10 (USP10) and ALK, leading to a decrease in the abundance of ALK protein. In summary, DAB2IP impairs the stability of ALK through USP10-dependent deubiquitination, suppressing Cortactin phosphorylation, thereby inhibiting invadopodia formation and metastasis of breast cancer cells. Furthermore, this study suggests a potential therapeutic strategy for breast cancer treatment.

Yes-associated protein 1 (YAP1), a central component of the Hippo pathway, plays an important role in tumor metastasis; however, the underlying mechanism remains to be elucidated. Invadopodia are actin-rich protrusions containing multiple proteases and have been widely reported to promote cell invasiveness by degrading the extracellular matrix. In the present study, we report that YAP1 induces invadopodia formation and promotes tumor metastasis in breast cancer cells. We also identify TIAM1, a guanine nucleotide exchange factor, as a target of the YAP1-TEAD4 complex. Our results demonstrate that YAP1 could promote TEAD4 binding to the enhancer region of TIAM1, which activates TIAM1 expression, subsequently increasing RAC1 activity and inducing invadopodia formation. These findings reveal the functional role of Hippo signaling in the regulation of invadopodia and provide potential molecular targets for preventing tumor metastasis in breast cancer.

Uneven oxygen supply in solid tumors leads to hypoxic and normoxic regions. Hypoxic cells exhibit increased secretion of lactate, which creates an acidic tumor microenvironment (TME). This acidic TME is positively associated with tumor metastasis. Despite the increased metastatic capacity of hypoxic cells, they are located relatively further away from the blood vessels and have limited access to the circulatory system. Studies have shown that cancer stem cells (CSCs) are enriched for tumor metastasis-initiating cells and generally undergo aerobic respiration, which could be enhanced by lactate. We therefore hypothesized that TME-derived lactate may promote the metastasis of normoxic CSCs. In the present study, the abundance of hypoxic and normoxic CSCs was analyzed in primary CRC tumors. It was found that the proportion of normoxic CSCs was positively associated with tumor stage. Using two human CRC cell lines, LoVo and SW480, and a patient-derived xenograft (XhCRC), it was found that treatment with lactate promoted normoxic CSC metastasis. Metabolism analysis indicated that, upon treatment with lactate, oxidative phosphorylation (OXPHOS) activity in normoxic CSCs was enhanced, whereas hypoxic CSCs were rarely altered. At the molecular level, the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of lactate oxidation, was found to be elevated in normoxic CSCs. Furthermore, PGC-1α knockdown markedly reduced the metastatic potential of normoxic CSCs. Notably, both the PGC-1α-mediated OXPHOS activity and metastatic potential were impaired when hypoxia-inducible factor-1α (HIF-1α) was activated in normoxic CSCs. Together, these findings provide a therapeutic strategy against tumor metastasis through the targeting of PGC-1α and, thus, the suppression of lactate-feeding OXPHOS in normoxic CSCs may improve the therapeutic benefit of patients with cancer, particularly CRC.

Tumor heterogeneity is an important feature of malignant tumors, and cell subpopulations may positively interact to facilitate tumor progression. Studies have shown that hypoxic cancer cells possess enhanced metastatic capacity. However, it is still unclear whether hypoxic cancer cells may promote the metastasis of normoxic cells, which have greater access to the blood circulation. When cocultured with hypoxic CRC cells or treated with hypoxic CRC cell-derived CM, normoxic CRC cells possessed increased metastatic capacity. Furthermore, hypoxic CRC cell-derived CM was enriched in interleukin 8. Hypoxic CRC cell-derived CM and recombinant human IL-8 both enhanced the metastatic capacity of normoxic cells by increasing the phosphorylation of p65 and then by inducing epithelial-mesenchymal transition. Knockdown of IL-8 in hypoxic CRC cells or the use of an anti-IL-8 antibody attenuated the CM- or rhIL-8-induced prometastatic capacity of normoxic CRC cells. Inhibition or knockdown of p65 abrogated IL-8-induced prometastatic effects. Most importantly, hypoxia-treated xenograft tumors enhanced the metastasis of normoxic CRC cells. Hypoxic CRC cell-derived IL-8 promotes the metastatic capacity of normoxic cells, and novel therapies targeting the positive interactions between hypoxic and normoxic cells should be developed.

This paper investigates the three-dimensional spatial characteristics of graphite in compacted graphite iron (CGI), at different crystallization stages, by interrupting the solidification process through experimental technique and taking three-dimensional X-ray diffraction (XRD) images through contrast tomography technique. The nucleation, growth mechanism and degeneration of graphite were analyzed by electron probe microanalysis technique (EPMA), scanning electron microscope with energy disperse spectroscopy (SEM-EDS), and transmission electron microscopes (TEM). The relationship between the maximum volume, connectivity and duration of crystallization was established by the analysis of the growth dynamics. The results have established a further relationship of nucleation between compacted graphite and spheroidal graphite. It was confirmed that the nucleation of graphite in CGI has a mechanism identical to that in the spherical graphite iron. The investigation proves that Ce2S3, La2S3, and Ti2S3 have nucleation capacity and that the growth mechanism of compacted graphite involves the evolution from spheroidal to cluster-like and then to interconnected graphite. The investigation also proves that the formation of compacted graphite is based on the graphite growth and graphite degeneration, which finally forms a worm-like morphology. According to the analysis of growth dynamics, the connectivity can be used as a key index for the CGI graphite nodularity and will affect the comprehensive properties of the material such as strength, thermal fatigue, thermal conductivity. The results are expected to provide a better understanding of compacted graphite iron as well as a better enrichment of the nucleation and growth mechanism of CGI.

BACKGROUND RNAi-based technology has achieved good results in both in vitro and in vivo applications, and it is expected to become a good genetic treatment for some diseases, especially neoplastic diseases. But there are still many obstacles in the in vivo application, the most important thing is the lack of an efficient and safe carrier. METHODS In this study, we designed and constructed a new siRNA delivery, which was named as aptamer-protamine-siRNA nanoparticle (APR). APR was consisted of ErbB3 aptamer, protamine and siRNA. We used Zeta nanosize to detect the size of APR to verify whether it is a nano-scale compound. We use the FAMRNA to replace the siRNA to detect whether APR could recognize and enter ErbB3 positive MCF-7 cells. Then we replaced the siRNA as oncogene suvivin siRNA to detect whether APR could inhibit tumor growth by silence surviving, and replaced siRNA to CDK1 siRNA to detect the cell cycle blocking effect. At last we tested the anticancer effect and safety of APR by carrying survivin siRNA in MCF-7 bearing nude mice. RESULTS APR was identified as a nanoscale compound. It showed specific targeting for ErbB3-positive MCF-7 cancer cells. APR has demonstrated the characteristics of inhibiting tumor growth by carrying siRNA against oncogene survivin. APR could also block cell cycle of MCF-7 cells by delivering CDK1 siRNAs. In the ErbB3 positive breast cancer xenograft mice model, APR nanoparticles could inhibit tumor growth and cause tumor regression without any toxicity. CONCLUSIONS In both in vivo and in vitro applications, APR nanoparticles could be targeted to recognize and enter ErbB3 positive tumor cells, and play a corresponding role by silencing targeted gene expression. APR nanoparticle is expected to become a good tumor treatment option.

Tumors harbor diverse compartments of cells with distinct metabolic properties and phenotypes, but the mechanism by which metabolic commensalism among distinct subsets of cancer cells affects tumor progression remains unclear. Colorectal cancer (CRC) has been reported to consist of cancer stem cells (CSCs) and differentiated cancer cells (non-CSCs). In the present study, organoid models were employed to show that CSCs and non-CSCs in CRC were characterized by distinct metabolic phenotypes. Treatment with either non-CSC-derived conditioned medium or exogenous lactate enhanced organoid-forming and tumor-initiating capacity of CSCs. In tumor regeneration assays with co-implanted CSCs and non-CSCs, the tumor-initiating activity was reduced when either monocarboxylate transporter (MCT)4 in non-CSCs or MCT1 in CSCs was silenced or inhibited. Mechanistically, oxiadative phosphorylation-derived reactive oxygen species in CSCs activated AKT-Wnt/β-catenin signaling, which could be induced by lactate from non-CSCs. Overall, these results suggest that CSCs and non-CSCs possess distinct metabolic profiles and, unexpectedly, non-CSC-originated lactate promotes self-renewal of CSCs and thus contributes to CRC progression. Our findings establish a rationale for developing novel therapies targeting the metabolic commensalism between different cell populations in CRC.

Three-dimensional (3D) cultures are indispensable for capturing tumor heterogeneity in colorectal cancer (CRC) in vitro. Although 3D cultures (such as sphere-forming assay and organoid culture) can partially preserve the morphological and molecular characteristics of primary CRC, whether these 3D cultures maintain the long-term stemness of cancer stem cells (CSCs) remains largely unknown. In the present study, spheres and organoids were generated side by side using individual primary CRC specimens, then respectively processed as serial passages. The results revealed that during serial passages, the percentage of CSCs (such as cluster of differentiation-133(+) and Wnt(+) cells) in organoids and the tumor-initiating capacity of organoid-derived cells were constant, while they gradually increased in the sphere-derived cells. Furthermore, during serial passages, resistance to chemotherapeutic agents (including 5-fluorouracil and oxaliplatin) in sphere- and organoid-derived cells was evaluated. The results indicated that the percentage of chemoresistant cells was constant in serial organoid cultures; however, it gradually increased in the serial sphere-forming assays. Taken together, the results of the present study comprehensively demonstrate that, with regard to long-term culture in vitro, organoid culture may be useful in maintaining tumor heterogeneity and the levels of chemoresistant cells, while the sphere formation assay enriches for CSCs and chemoresistant cells.

Objective To explore the role of Hypoxia inducible factor-1 α (HIF-1 α) on the proliferation of the colorectal cancer cells and the mechanism.Methods The expression of histone demethylase 4B (KDM4B) and HIF-1α was detected by Western blotting assay in normoxia and hypoxia;The cell proliferation activity was detected by cell counting kit-8 (CCK-8),and cell cycle was detected by Flow CytoMetry.The data were analyzed by statistical methods (P value less than 0.05 was considered statistically significant).Results The expression of HIF-1α was positively correlated with KDM4B.The Western blotting assay showed that expression of HIF-1α and KDM4B were significant higher in hypoxia model (H) than normoxia(N).CCK-8 showed that A value of siKDM4B group at 24 h (N:0.793 ±0.008,H:0.529 ± 0.021) was significantly different from that of Control Group (N:1.077 ± 0.017,H:0.826 ± 0.019,t =3.452,P ＜ 0.05).At 48 h and 72 h,the A value of the experimental group was lower than control group (P ＜ 0.05).Flow CytoMetry showed that S phase cell proportion of the siKDM4B group (N:40.64％,H:35.50％) was significantly lower than control group (N:56.68％,H:45.64％) (P ＜0.05).Conclusion HIF-1α has a vital role in proliferation activity by regulating KDM4B in colorectal cancer cells.

BACKGROUND:Focal adhesion plays an essential role in tumour invasiveness and metastasis. Hippo component YAP has been widely reported to be involved in many aspects of tumour biology. However, its role in focal adhesion regulation in breast cancer remains unexplored.METHODS:Tissue microarray was used to evaluate YAP expression in clinical breast cancer specimens by immunohistochemical staining. Cell migration and invasion abilities were measured by Transwell assay. A cell adhesion assay was used to measure the ability of cell adhesion to gelatin. The focal adhesion was visualized through immunofluorescence. Phosphorylated FAK and other proteins were detected by Western blot analysis. Gene expression profiling was used to screen differently expressed genes, and gene ontology enrichment was performed using DAVID software. The gene mRNA levels were measured by quantitative real-time PCR. The activity of the THBS1-promoter was evaluated by dual luciferase assay. Chromatin immunoprecipitation (ChIP) was used to verify whether YAP could bind to the THBS1-promoter region. The prediction of potential protein-interaction was performed with the String program. The ChIP sequence data of TEAD was obtained from the ENCODE database and analysed via the ChIP-seek tool. The gene expression dataset (GSE30480) of purified tumour cells from primary breast tumour tissues and metastatic lymph nodes was used in the gene set enrichment analysis. Prognostic analysis of the TCGA dataset was performed by the SurvExpress program. Gene expression correlation of the TCGA dataset was analysed via R2: Genomics Analysis and Visualization Platform.RESULTS:Our study provides evidence that YAP acts as a promoter of focal adhesion and tumour invasiveness via regulating FAK phosphorylation in breast cancer. Further experiments reveal that YAP could induce FAK phosphorylation through a TEAD-dependent manner. Using gene expression profiling and bioinformatics analysis, we identify the FAK upstream gene, thrombospondin 1, as a direct transcriptional target of YAP-TEAD. Silencing THBS1 could reverse the YAP-induced FAK activation and focal adhesion.CONCLUSION:Our results unveil a new signal axis, YAP/THBS1/FAK, in the modulation of cell adhesion and invasiveness, and provides new insights into the crosstalk between Hippo signalling and focal adhesion.

Cancer stem cells (CSCs) are inherently resistant to chemotherapy, and CSCs in chemotherapy-failed recurrent tumors are enriched; however, the cellular origin of chemotherapy-induced CSC enrichment remains unclear. Communication with stromal fibroblasts may induce cancer cell dedifferentiation into CSCs through secreted factors. We recently demonstrated that fibroblast-derived exosomes promote chemoresistance in colorectal cancer (CRC). Here, we report that fibroblasts confer CRC chemoresistance via exosome-induced reprogramming (dedifferentiation) of bulk CRC cells to phenotypic and functional CSCs. At the molecular level, we provided evidence that the major reprogramming regulators in fibroblast-exosomes are Wnts. Exosomal Wnts were found to increase Wnt activity and drug resistance in differentiated CRC cells, and inhibiting Wnt release diminished this effect in vitro and in vivo. Together, our results indicate that exosomal Wnts derived from fibroblasts could induce the dedifferentiation of cancer cells to promote chemoresistance in CRC, and suggest that interfering with exosomal Wnt signaling may help to improve chemosensitivity and the therapeutic window.

Objective The aim of the study was to investigate whether colon cancer stem cells induced by epidermal growth factor (EGF) to enter the cell cycle enhanced the chemosensitivity of colon cancer. Methods In vitro , EGF was used to stimulate the entry of human colon cancer HCT116 cells into the cell cycle. Before and after treatment with EGF, CD133+ HCT116 cells were collected and flow cytometry was conducted to determine the apoptosis rate based on the 5-Fu and Ki-67 expression rates. The cell cycle distribution of the two groups was also determined. In vivo , a subcutaneous xenograft model of HCT116 human colon cancer cell lines in nude mice was established. The nude mice were divided into two groups and treated with EGF and 5-Fu, respectively. Differences in the growth of implanted tumors revealed the efficiency of cycle-induction combined chemotherapy. Results (1) After EGF stimulation, the S-G2/M proportion of CD133+ HCT116 cells and Ki67 expression were increased, indicating that more cancer stem cells entered the cell cycle and promoted proliferation; (2) After EGF stimulation, CD133+ HCT116 cells showed a higher apoptosis rate induced by 5-Fu. (3) Animal experiments showed that the group subjected to combined treatment with EGF and 5-Fu had smaller tumor sizes compared to the group treated with 5-Fu alone. Conclusion EGF enhanced tumor sensitivity to chemotherapeutic drugs, likely by promoting tumor stem cells to enter the cell cycle.

7544 Background: The aim of the study was to analyze the driver mutation profiles in a large cohort of Chinese SqCLCs to identify potential therapeutic targets. Methods: We detected approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes on 159 samples by using Ion Torrent semiconductor-based next-generation sequencing.We conducted FISH for fibroblast growth factor receptor 1 (FGFR1) and cyclin D1 (CCND1) amplification on 172 and 246 samples, respectively. In addtion, we analyzed PTEN expression by immunohistochemistry on 172 samples. Results: Somatic mutations were detected in 73.6% (117/159) of patients. The most commonly mutated gene detected in this study was TP53 (89 cases, 56.0%), followed by CDKN2A (14 cases, 8.8%), PI3KCA (14 cases, 8.8%), KRAS (7 cases, 4.4%), and EGFR (5 cases, 3.1%). The incidences of FGFR1 amplification, CCND1 amplification and loss of PTEN expression in this cohort of patients were 16.9% (29/172), 11% (27/246) and 43.6% (75/172), respectively. The fr...

Colorectal cancer (CRC) is heterogeneous and contains different-sized cells. Recent studies have shown that tumor-initiating cells (TICs) are involved in cancer initiation, recurrence and metastasis. However, connections between cancer cell size and stem-like properties are largely unknown. Here we purified large- and small-sized CRC cells by fluorescence-activated cell sorting (FACS) based on forward scatter (FSC), and demonstrated that small CRC cells possess higher holoclone- and sphere-forming capacity in vitro, tumor-initiating capacity in vivo and form more lung metastases compared with large CRC cells. Furthermore, we found that down-regulated YAP1 (yes-associated protein 1) decreased tumor-initiating and metastatic capacity in small CRC cells but not in large CRC cells. More importantly, our results showed that the expression of YAP1 positively correlated with the poor prognosis in CRCs. Collectively, our findings suggest that small CRC cells enrich for metastatic TICs, and YAP1 is one of the potential therapeutic targets of metastatic TICs, the small CRC cells.

Serum carcinoembryonic antigen (CEA) is the most commonly used tumor marker in a variety of cancers including colorectal cancer (CRC) for tumor diagnosis and monitoring. Recent studies have shown that colonic crypt cells expressing little or no CEA may enrich for stem cells. Numerous studies have clearly shown that there exist CRC patients with normal serum CEA levels during tumor progression or even tumor relapse, although CEA itself is considered to promote metastasis and block cell differentiation. These seemingly contradictory observations prompted us to investigate, herein, the biological properties as well as tumorigenic and metastatic capacity of CRC cells that express high (CEA+) versus low CEA (CEA-(/lo)) levels of CEA. Our findings show that the abundance of CEA-/lo cells correlate with poor differentiation and poor prognosis, and moreover, CEA-/lo cells form more spheres in vitro, generate more tumors and exhibit a higher potential in developing liver and lung metastases than corresponding CEA+ cells. Applying RNAi-mediated approach, we found that IGF1R mediated tumorigenic and capacity of CEA-(/lo) cells but did not mediate those of CEA+ cells. Notably, our data demonstrated that CEA molecule was capable of protecting CEA-(/lo) cells from anoikis, implying that CEA+ cells, although themselves possessing less tumorigenic and metastatic capacity, may promote metastasis of CEA-(/lo) cells via secreting CEA molecule. Our observations suggest that, besides targeting CEA molecule, CEA-(/lo) cells may represent a critical source of tumor progression and metastasis, and should therefore be the target of future therapies.