Invasive Cell Phenotype Research Articles

Heterogeneity of the Alpha-Smooth Muscle Actin Tumor Score in Breast Cancer Cells Significantly Affects Tumor Invasiveness, Recurrence, and Patient Survival.

Alpha-smooth muscle actin (αSMA) has been widely investigated in malignancies, primarily concerning its expression in cancer-associated fibroblasts (CAFs) inside the tumor stroma. Microscopic examination indicates that αSMA expression is not confined to the tumor stromal compartment but is also present in a subset of tumor cells, and this expression correlates with an enhanced invasive phenotype of malignant cells from lung, liver, or ovarian malignancies. Information on actin expression in breast cancer (BC) cells is scarce, and its influence on clinicopathological characteristics remains ambiguous due to conflicting findings in the literature. To examine the αSMA tumor score in breast cancer cells utilizing digital image analysis (DIA) methodologies and to critically analyze the varying effects of αSMA tumor score values on clinicopathologic parameters, particularly focusing on tumor cell invasiveness, recurrence, and survival. Double immunostaining for CD34 and αSMA was conducted on 53 breast cancer cases that were thoroughly characterized in relation to clinicopathologic data. Double immunostaining for CD34 and αSMA demonstrated different distribution patterns of both markers in normal and breast cancer tissues. DIAdata about αSMA tumor cell density, intensity, tumor score, and histological score were correlated with clinicopathological factors. We delineated three unique breast cancer subgroups based on αSMA tumor scores: a 9.43% low-expressing subgroup (αSMA_TSlow, score 4), a 35.07% medium-expressing subgroup (αSMA_TSmed, scores 5 and 6), and a 55.5% high-expressing subgroup (αSMA_TShigh, scores 7 and 8). Stromal immature vessels and tertiary lymphoid structures (TLS) exhibited a strong correlation with αSMA_TSlow, whereas recurrence, perineural, and lymphovascular invasion strongly influenced the αSMA_TSmed and αSMA_TShigh subgroups. The αSMA_TSmed subgroup demonstrated the most heterogeneity with the influence of αSMA-expressing breast cancer cells on tumor size, nodal status, perineural and lymphovascular invasion, menopausal status, recurrence, and survival. Most of the cases from the αSMA_TSlow subgroup had Luminal B and Luminal B-HER2 phenotypes, while triple-negative breast cancer (TNBC) represented one-third of all cases in the αSMA_TShigh subgroup. αSMA-expressing breast cancer cells variably affect malignant growth, invasion, and recurrence, highly contingent upon their density and expression intensity. The current investigation identified an αSMA_TSmed BC subgroup that appears to promote invasiveness, recurrence, and survival in breast cancer. Our data indicate that αSMA BC-expressing cells play a dual role in BC progression, contingent upon their percentage and expression intensity; however, further research is required to elucidate the factors and mechanisms responsible for their accumulation and/or transdifferentiation in malignant breast tissue.

Abstract PR003: Sex-dependent changes in the aged melanoma tumor microenvironment influence metastasis and therapeutic responses

Abstract There is documented sex disparity in cutaneous melanoma incidence and mortality, increasing disproportionately with age and in the male sex. However, the underlying mechanisms remain unclear; while biological sex differences and inherent immune response variability have been assessed in tumor cells, the role of the tumor-surrounding microenvironment, contextually in aging, has been overlooked. Dermal fibroblasts are known to have profound impact on melanoma progression. We examined if both sex-dependent and age-related changes in these fibroblasts can alter the course of melanoma tumor growth, visceral metastasis, and responses to therapy. We find that skin fibroblasts undergo age-mediated sex-differential changes in their proliferation, senescence, ROS production and stress response. We find that aged male fibroblasts drive an invasive, therapy-resistant phenotype in melanoma cells and promote metastasis in aged male mice by increasing AXL expression. Intrinsic aging promotes sex- dimorphic BMP2 secretion exclusively by aged male fibroblasts, which in turn drives the slower-cycling, highly invasive, and therapy-resistant melanoma cell phenotype, characteristic of the aged male TME. Inhibition of BMP2 activity blocks the emergence of invasive phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. Our data provide an integrated view of how host factors such as advancing age and biological sex alter the tumor microenvironment and contribute to disease progression. Bridging this knowledge gap will improve patient stratification and assist in tailoring the therapy and achieve gender-equitable healthcare. Citation Format: Yash Chhabra, Ashani T. Weeraratna. Sex-dependent changes in the aged melanoma tumor microenvironment influence metastasis and therapeutic responses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR003.

CircSEC24B activates autophagy and induces chemoresistance of colorectal cancer via OTUB1-mediated deubiquitination of SRPX2

Circular RNAs (circRNAs) are a type of regulatory RNA that feature covalently closed single-stranded loops. Evidence suggested that circRNAs play important roles in the progression and development of various cancers. However, the impact of circRNA on autophagy-mediated progression of colorectal cancer (CRC) remains unclear. The objective of this project was to investigate the influence of circSEC24B on autophagy and its underlying mechanisms in CRC. To validate the presence and circular structure of circSEC24B in CRC cells and tissues, PCR and Sanger sequencing techniques were employed. Drug resistance and invasive phenotype of CRC cells were evaluated using CCK8, transwell, and Edu assays. Gain- and loss-of-function experiments were conducted to assess the effects of circSEC24B and its protein partner on the growth, invasion, and metastasis of CRC cells in vitro and in vivo. Interactions between circSEC24B, OTUB1, and SRPX2 were analyzed through immunofluorescence, RNA-pulldown, and RIP assays. Mass spectrometry analysis was used to identify potential binding proteins of circRNA in CRC cells. Vectors were constructed to investigate the specific structural domain of the deubiquitinating enzyme OTUB1 that binds to circSEC24B. Results showed that circSEC24B expression was increased in CRC tissues and cell lines, and it enhanced CRC cell proliferation and autophagy levels. Mechanistically, circSEC24B promoted CRC cell proliferation by regulating the protein stability of SRPX2. Specifically, circSEC24B acted as a scaffold, facilitating the binding of OTUB1 to SRPX2 and thereby enhancing its protein stability. Additionally, evidence suggested that OTUB1 regulated SRPX2 expression through an acetylation-dependent mechanism. In conclusion, this study demonstrated that circSEC24B activated autophagy and induced chemoresistance in CRC by promoting the deubiquitination of SRPX2, mediated by the deubiquitinating enzyme OTUB1.

Sex-dependent effects in the aged melanoma tumor microenvironment influence invasion and resistance to targeted therapy

There is documented sex disparity in cutaneous melanoma incidence and mortality, increasing disproportionately with age and in the male sex. However, the underlying mechanisms remain unclear. While biological sex differences and inherent immune response variability have been assessed in tumor cells, the role of the tumor-surrounding microenvironment, contextually in aging, has been overlooked. Here, we show that skin fibroblasts undergo age-mediated, sex-dependent changes in their proliferation, senescence, ROS levels, and stress response. We find that aged male fibroblasts selectively drive an invasive, therapy-resistant phenotype in melanoma cells and promote metastasis in aged male mice by increasing AXL expression. Intrinsic aging in male fibroblasts mediated by EZH2 decline increases BMP2 secretion, which in turn drives the slower-cycling, highly invasive, and therapy-resistant melanoma cell phenotype, characteristic of the aged male TME. Inhibition of BMP2 activity blocks the emergence of invasive phenotypes and sensitizes melanoma cells to BRAF/MEK inhibition.

Investigating the effect of Fusobacterium nucleatum on the aggressive behavior of cancer-associated fibroblasts in colorectal cancer

Fusobacterium nucleatum, (F. nucleatum) as a known factor in inducing oncogenic, invasive, and inflammatory responses, can lead to an increase in the incidence and progression of colorectal cancer (CRC). Cancer-associated fibroblasts (CAF) are also one of the key components of the tumor microenvironment (TME), which lead to resistance to treatment, metastasis, and disease recurrence with their markers, secretions, and functions. This study aimed to investigate the effect of F. nucleatum on the invasive phenotype and function of fibroblast cells isolated from normal and cancerous colorectal tissue. F. nucleatum bacteria were isolated from deep periodontal pockets and confirmed by various tests. CAF cells from tumor tissue and normal fibroblasts (NF) from a distance of 10 cm of tumor tissue were isolated from 5 patients by the explant method and were exposed to secretions and ghosts of F. nucleatum. The expression level of two markers, fibroblast activation protein (FAP), and α-smooth muscle actin (α-SMA), and the amount of production of two cytokines TGF-β and IL-6 from fibroblast cells were measured by flow cytometry and ELISA test, respectively before and after exposure to different bacterial components. The expression of the FAP marker was significantly higher in CAF cells compared to NF cells (P < 0.05). Also, the expression of IL-6 in CAF cells was higher than that of NF cells. In investigating the effect of bacterial components on the function of fibroblastic cells, after comparing the amount of IL-6 produced between the normal tissue of each patient and his tumoral tissue under 4 treated conditions, it was found that the amount of IL-6 production from the CAF cells of patients in the control group, treated with heat-killed ghosts and treated with paraformaldehyde-fixed ghosts had a significant increase compared to NF cells (P < 0.05). Due to the significant increase in FAP marker expression in fibroblast cells of tumor tissue compared to normal tissue, it seems that FAP can be used as a very good therapeutic marker, especially in patients with high levels of CAF cells. Various components of F. nucleatum could affect fibroblast cells differentially and at least part of the effect of this bacterium in the TME is mediated by CAF cells.

Mammary adipocytes promote breast tumor cell invasion and angiogenesis in the context of menopause and obesity

The mechanism(s) underlying obesity-related postmenopausal (PM) breast cancer (BC) are not clearly understood. We hypothesized that the increased local presence of ‘obese’ mammary adipocytes within the BC microenvironment promotes the acquisition of an invasive and angiogenic BC cell phenotype and accelerates tumor proliferation and progression. BC cells, treated with primary mammary adipocyte secretome from premenopausal (Pre-M) and PM obese women (ObAdCM; obese adipocyte conditioned-media) upregulated the expression of several pro-tumorigenic factors including VEGF, lipocalin-2 and IL-6. Both Pre-M and PM ObAdCM stimulated endothelial cell recruitment and proliferation and significantly stimulated BC cell proliferation, migration and invasion. IL-6 and LCN2 induced STAT3/Akt signaling in BC cells and STAT3 inhibition abrogated the ObAdCM-stimulated BC cell proliferation and migration. Expression of proangiogenic regulators including VEGF, NRP1, NRP2, IL8RB, TGFβ2, and TSP-1 were found to be differentially regulated in mammary adipocytes from obese PM women. Comparative RNAseq indicated an upregulation of PI3K/Akt signaling, ECM-receptor interactions and lipid/fatty acid metabolism in PM versus Pre-M mammary adipocytes. Our results demonstrate that irrespective of menopausal status, cross-talk between obese mammary adipocytes and BC cells promotes tumor aggressiveness and suggest that targeting the LCN2/IL-6/STAT3 signaling axis may be a useful strategy in obesity-driven breast tumorigenesis.

The potential mechanism of WT1-associated protein-induced N-6-methyladenosine modification of colony-stimulating factor 2 in the progression of oral squamous cell carcinoma by JAK/STAT3 pathway regulation.

Colony-stimulating factor 2 (CSF2) plays a regulatory role in numerous cancers. However, there is needed to investigate the role of CSF2 in oral squamous cell carcinoma (OSCC) malignant phenotype and the specific mechanisms of CSF2 N-6-methyladenosine (m6A) modification. Therefore, we investigated the regulatory mechanism of m6A-modified CSF2 by WT1-associated protein (WTAP) in OSCC via qRT-PCR, western blot, WTAP and CSF2 overexpression in OSCC. In a panel of OSCCs, Kaplan-Meier plot analysis indicated that high expression of CSF2 was associated with poorer prognosis. Cell functional experiments revealed that enrichment of CSF2 promoted the proliferation and migration of OSCC cells by activating the JAK/STAT3 pathway, whereas the reduced expression of CSF2 resulted in the malignant decline of OSCC cells by blocking the JAK/STAT3 pathway. This study also confirmed that WTAP enhanced the m6A level of CSF2 and facilitated the expression of CSF2 and that CSF2 silencing blocked the invasive phenotype of OSCC cells and reversed the malignancy induced by WTAP overexpression. Overall, this study demonstrated that WTAP mediates the m6A modification of CSF2 and the JAK/STAT3 pathway, which plays an oncogenic role in the development of OSCC and can be a target for the treatment of patients with OSCC.

Biomimetic “nano-spears” for CAFs-targeting: splintered three “shields” with enhanced cisplatin anti-TNBC efficiency

The treatment dilemma of triple-negative breast cancer (TNBC) revolves around drug resistance and metastasis. Cancer-associated fibroblasts (CAFs) contribute to cisplatin (Cis) resistance and further metastasis in TNBC, making TNBC a difficult-to-treat disease. The dense stromal barrier which restricts drug delivery, invasive phenotype of tumor cells, and immunosuppressive tumor microenvironment (TME) induced by CAFs serve as three “shields” for TNBC against Cis therapy. Here, we designed a silybin-loaded biomimetic nanoparticle coated with anisamide-modified red blood cell membrane (ARm@SNP) as a “nanospear” for CAFs-targeting, which could shatter the “shields” and significantly exhibit inhibitory effect on 4T1 cells in combination with Cis both in vitro and in vivo. The ARm@SNP/Cis elicited 4T1 tumor growth arrest and destroyed three “shields” as follows: disintegrating the stromal barrier by inhibiting blood vessels growth and the expression of fibronectin; decreasing 4T1 cell invasion and metastasis by affecting the TGF-β/Twist/EMT pathway which impeded EMT activation; reversing the immunosuppressive microenvironment by increasing the activity and infiltration of immunocompetent cells. Based on CAFs-targeting, ARm@SNP reversed the resistance of Cis, remodeled the TME and inhibited invasion and metastasis while significantly improving the therapeutic effect of Cis on 4T1 tumor-bearing mice, providing a promising approach for treating intractable TNBC.

The role of CXCL2-mediated crosstalk between tumor cells and macrophages in Fusobacterium nucleatum-promoted oral squamous cell carcinoma progression

Dysbiosis of the oral microbiota is related to chronic inflammation and carcinogenesis. Fusobacterium nucleatum (Fn), a significant component of the oral microbiota, can perturb the immune system and form an inflammatory microenvironment for promoting the occurrence and progression of oral squamous cell carcinoma (OSCC). However, the underlying mechanisms remain elusive. Here, we investigated the impacts of Fn on OSCC cells and the crosstalk between OSCC cells and macrophages. 16 s rDNA sequencing and fluorescence in situ hybridization verified that Fn was notably enriched in clinical OSCC tissues compared to paracancerous tissues. The conditioned medium co-culture model validated that Fn and macrophages exhibited tumor-promoting properties by facilitating OSCC cell proliferation, migration, and invasion. Besides, Fn and OSCC cells can recruit macrophages and facilitate their M2 polarization. This crosstalk between OSCC cells and macrophages was further enhanced by Fn, thereby amplifying this positive feedback loop between them. The production of CXCL2 in response to Fn stimulation was a significant mediator. Suppression of CXCL2 in OSCC cells weakened Fn’s promoting effects on OSCC cell proliferation, migration, macrophage recruitment, and M2 polarization. Conversely, knocking down CXCL2 in macrophages reversed the Fn-induced feedback effect of macrophages on the highly invasive phenotype of OSCC cells. Mechanistically, Fn activated the NF-κB pathway in both OSCC cells and macrophages, leading to the upregulation of CXCL2 expression. In addition, the SCC7 subcutaneous tumor-bearing model in C3H mice also substantiated Fn’s ability to enhance tumor progression by facilitating cell proliferation, activating NF-κB signaling, up-regulating CXCL2 expression, and inducing M2 macrophage infiltration. However, these effects were reversed by the CXCL2-CXCR2 inhibitor SB225002. In summary, this study suggests that Fn contributes to OSCC progression by promoting tumor cell proliferation, macrophage recruitment, and M2 polarization. Simultaneously, the enhanced CXCL2-mediated crosstalk between OSCC cells and macrophages plays a vital role in the pro-cancer effect of Fn.

Abstract 5513: Sexual differences in the aged melanoma tumor microenvironment dictates metastasis and therapeutic responses

Abstract There is documented sex disparity in cutaneous melanoma incidence and mortality, increasing disproportionately with age and in the male sex. However, the underlying mechanisms remain unclear; while biological sex differences and inherent immune response variability have been assessed in tumor cells, the role of the microenvironment surrounding the tumor, contextually in aging, has been overlooked. We find that skin fibroblasts undergo age-mediated changes in their proliferation, senescence, ROS levels and stress response that vary with sex. We find that aged male fibroblasts selectively drive an invasive and slow-cycling phenotype in melanoma cells in vitro by increasing AXL expression. This is also evident in syngeneic mouse models where metastasis is increased in aged male mice. Mechanistically, intrinsic aging in male fibroblasts coupled with elevated ROS promotes EZH2 decline thereby increasing BMP2 secretion, which in turn drives the slower-cycling, highly invasive, and therapy-resistant melanoma cell phenotype, characteristic of the aged male TME. Inhibition of BMP2 activity blocks the emergence of the invasive phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. Our data provides an integrated view of how age and sex of the host contributes to melanoma progression and therapy responses. Bridging this knowledge gap will improve patient stratification and assist in tailoring the therapy to the individual. Citation Format: Yash Chhabra, Mitchell Fane, Sneha Pramod, Laura Hueser, Daniel Zabransky, Vania Wang, Edwin Kumah, Alexis Carey, Elizabeth Harper, Murilo Ramos Rocha, Ashani Weeraratna. Sexual differences in the aged melanoma tumor microenvironment dictates metastasis and therapeutic responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5513.

Abstract 5168: Analysis of biomarker and outcomes data: 7-year update from COLUMBUS

Abstract Background: In the randomized, 2-part, phase 3 COLUMBUS study (NCT01909453), encorafenib (enco) + binimetinib (bini)—an approved regimen in the US, EU, and other countries—improved 7-year PFS and OS vs vemurafenib (vemu) in patients (pts) with BRAF V600E/K-mutant locally advanced unresectable or metastatic melanoma. We investigated genetic and transcriptional correlates of outcomes in an exploratory biomarker analysis of COLUMBUS Parts 1 and 2. Methods: In Part 1, 577 pts were randomized 1:1:1 to enco 450 mg QD + bini 45 mg BID, enco 300 mg QD, or vemu 960 mg BID; in Part 2, 344 pts were randomized 3:1 to enco 300 mg QD + bini 45 mg BID or enco 300 mg QD. Baseline tumor samples were analyzed using the ACE ImmunoID NeXT whole exome sequencing/whole transcriptome sequencing (WES/WTS) assays (Personalis). PFS and OS were analyzed (cutoff: Jan 13, 2023) by treatment arm and presence of specific genetic or transcriptomic alterations. Results: Among treated pts, 668 had WES or WTS data. K-means clustering identified 3 major transcriptional clusters: (1) microphthalmia-associated transcription factor (MITF)-low (invasive dedifferentiated tumor cell phenotype), (2) MITF-high (proliferative melanocytic phenotype), and (3) immune-high. Cluster assignments were significantly associated with biopsy site (P&amp;lt;0.001), with cluster 1 enriched in skin biopsies and cluster 3 in lymph node metastases; biopsy site-independent characteristics were also captured. Improved PFS with enco + bini vs vemu was most pronounced in cluster 3 (Table 1). ERBB2 expression and PI3KCA pathway mutation, previously found to be associated with reduced survival benefit with enco + bini vs vemu, were enriched in clusters 1 and 2 vs cluster 3. Conclusions: Enco + bini showed benefit across molecular subtypes in BRAF V600E/K-mutant melanoma. The contribution of bini was most pronounced in the immune-high cluster. Improved outcomes in this cluster support ongoing trials of combinations with immune checkpoint inhibitors, such as STARBOARD and PORTSIDE. Table 1. Hazard ratios of PFS and OS for enco + bini or enco vs vemu by cluster HR (95% CI) vs vemu PFS OS Enco + bini Enco Enco + bini Enco Overall n=255 vs 100 n=159 vs 100 n=255 vs 100 n=159 vs 100 0.47 (0.35, 0.63) 0.78 (0.57, 1.07) 0.66 (0.50, 0.86) 0.85 (0.63, 1.13) Clusters Cluster 1: MITF-low n=72 vs 34 n=44 vs 34 n=72 vs 34 n=44 vs 34 0.74 (0.44, 1.26) 0.92 (0.51, 1.66) 0.63 (0.40, 1.00) 0.57 (0.34, 0.96) Cluster 2: MITF-high n=120 vs 46 n=76 vs 46 n=120 vs 46 n=76 vs 46 0.48 (0.32, 0.74) 0.75 (0.48, 1.17) 0.66 (0.45, 0.97) 0.86 (0.56, 1.32) Cluster 3: Immune-high n=63 vs 20 n=39 vs 20 n=63 vs 20 n=39 vs 20 0.23 (0.12, 0.44) 0.64 (0.33, 1.21) 0.80 (0.40, 1.59) 1.55 (0.76, 3.15) Numbers are given as the number of pts included in the treatment arm vs the number of pts in the comparator arm (vemu-treated). Statistical significance (P&amp;lt;0.05) is shown by bold font. Bini, binimetinib; enco, encorafenib; MITF, microphthalmia-associated transcription factor; pts, patients; vemu, vemurafenib. Citation Format: Reinhard Dummer, Keith Flaherty, Shibing Deng, Tao Xie, Phineas Hamilton, Nuzhat Pathan, Caroline Robert, Ana Arance, Jan Willem de Groot, Claus Garbe, Helen J. Gogas, Ralf Gutzmer, Ivana Krajsová, Gabriella Liszkay, Carmen Loquai, Mario Mandala, Dirk Schadendorf, Naoya Yamazaki, Paolo A. Ascierto, Alessandra di Pietro, Craig B. Davis. Analysis of biomarker and outcomes data: 7-year update from COLUMBUS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5168.

Abstract 284: Crosstalk with postmenopausal obese mammary adipocytes impacts breast cancer cell metabolism and promotes tumorigenesis

Abstract Introduction: Obesity is associated with an increased risk and a poor prognosis of breast cancer (BC) in postmenopausal (PM) women. The mechanism(s) underlying obesity-related PM BC are not clearly understood. We hypothesized that the increased local presence of ‘obese’ mammary adipocytes within the breast tumor microenvironment (TME) promotes the acquisition of an invasive BC cell phenotype and markedly accelerates tumor proliferation and progression. Methods: We asked whether cancer-associated mammary adipocytes (CA-MAd) differ from normal mammary adipocytes (N-MAd) in their ability to promote tumorigenesis of dormant BC cells and if so, what the underlying mechanism(s) might be. Using a 2D transwell model, BC cells (ER- and ER+), were cocultured with obese PM CA-MAd isolated from breast cancer patients or obese mammary N-MAds from PM disease-free women and analyzed for migration, proliferation, phenotypic, signaling and metabolic alterations. Results: Compared to monoculture (MC) conditions, BC cells cocultured (CC) with CA-MAd displayed significantly increased migration and proliferation. Quantitative phase imaging indicated that CA-MAd secretome-treated BC cells displayed increased motility, speed, distance covered and cell size/area compared to N-MAd. Consistent with these results, mesenchymal markers such as fibronectin, vimentin and SNAIL were upregulated whereas the epithelial marker E-cadherin was downregulated in CC vs. MC conditions suggesting that Ads in the TME promote EMT in BC cells. Interestingly, mature Ads CC with BC cells underwent delipidation and dedifferentiation as indicated by reduced Oil Red staining, decreased lipid content and reduced expression of Ad maturation markers such as FABP4, ADIPOQ and PLIN1. Consistently, we determined that CC BC cells exhibited increased lipid uptake and BODIPY staining and upregulated pAMPK, pACC and CPT1A signaling when compared to MC BC cells. Short term treatment of BC cells with both N-MAd and CA-MAd secretomes resulted in increased oxygen consumption rate (OCR), basal respiration, ATP-linked respiration and proton-linked respiration compared to control and as determined by the Seahorse extracellular flux assay. CC with MAds significantly downregulated OCR and all respiration parameters in BC cells suggesting decreased glucose uptake and glycolytic flux. Conclusions: Compared to MC, CC with obese M-Ads promoted functional, phenotypic and metabolic changes in BC cells. Taken together, our results indicate that crosstalk between BC cells and obese MAds promotes delipidation of MAds and a concomitant lipid uptake in BC cells and a switch to lipid metabolism signaling pathways that may, in turn, drive a metabolic shift in BC cells and promote tumor growth. [Supported by NIH RO1CA185530, the Breast Cancer Research Foundation, the Karp Family Foundation and the Nile Albright Research Foundation] Citation Format: Roopali Roy, Rama Aldakhlallah, Stephen Cobbs, Emily Man, Caleb Hallinan, Kwonmoo Lee, Margaret Lotz-Bousvaros, Susan Pories, Marsha A. Moses. Crosstalk with postmenopausal obese mammary adipocytes impacts breast cancer cell metabolism and promotes tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 284.

YAP1 controls the N-cadherin-mediated tumor-stroma interaction in melanoma progression

The hallmark of epithelial-to-mesenchymal transition (EMT) is the switch from epithelial cadherin (E-cadherin) to neural cadherin (N-cadherin), allowing melanoma cells to form a homotypic N-cadherin-mediated adhesion with stromal fibroblasts. However, how cadherin switching is initiated, maintained, and regulated in melanoma remains elusive. Here, we report a novel mechanism underlying cadherin switching in melanoma cells that is regulated by stromal Yes-associated protein 1 (YAP1) signaling. The progression of a BRAF-mutant mouse melanoma was suppressed in vivo upon YAP1 ablation in cancer-associated fibroblasts (CAFs). On the contrary, overexpressing YAP1 in CAFs accelerated melanoma development. By RNA-Seq, N-cadherin was identified as a major downstream effector of YAP1 signaling in CAFs. YAP1 silencing reduced N-cadherin expression in CAFs, leading to the downregulation of N-cadherin in neighboring melanoma cells. N-cadherin ablation inhibited the PI3K-AKT signaling pathway in melanoma cells and melanoma cell proliferation. The findings suggest that YAP1 depletion in CAFs induces the downregulation of p-AKT signaling in melanoma cells through the N-cadherin-mediated interaction between melanoma cells and CAFs. The data underscore an important role of CAFs in regulating N-cadherin-mediated adhesion and signaling in melanoma and highlight that disentangling cadherin-mediated cell-cell interactions can potentially disrupt tumor-stroma interactions and reverse the tumor cell invasive phenotype.

Baicalin induces ferroptosis in oral squamous cell carcinoma by suppressing the activity of FTH1.

This study investigated the role of the ferroptosis-related gene FTH1 in oral squamous cell carcinoma (OSCC) and evaluated the therapeutic potential of baicalin in OSCC cell treatment. A prognostic model was established by bioinformatic analysis, consisting of 12 ferroptosis related genes (FRGs), and FTH1 was selected as the most significantly up-regulated FRGs. The clinical correlation of FTH1 in OSCC samples was evaluated by both immunohistochemical and bioinformatic characterizations. The effects of FTH1 on migration, invasion, epithelial-mesenchymal transition (EMT) and proliferation were determined by wound healing assays, transwell assays, western blotting and 5'-ethynl 2'-deoxyuridine proliferation assays, respectively. The effects of FTH1 on ferroptosis were tested via ferroptosis markers and Mito Tracker staining. In addition, the therapeutic effects of baicalin on OSCC cells were confirmed using EMT, migration, invasion, proliferation and ferroptosis assays. The 12 FRGs werepredictive of theprognosis for OSCC patients, and FTH1 expression was identified as significantly up-regulated in OSCC samples, which was highly associated with survival, immune cell infiltration and drug sensitivity. Moreover, knocking down FTH1 inhibited cell proliferation, EMT and invasive phenotypes, but induced ferroptosis in OSCC cells (Cal27 and SCC25). Furthermore, baicalin directly suppressed expression of FTH1 in OSCC cells, and effectively promoted ferroptosis and inhibited the proliferation as well as EMT by directly targeting FTH1. This study has demonstrated that FTH1 is a therapeutic target for OSCC treatment, and has provided evidence that baicalin offers a promising alternative for OSCC treatment.

Sphingosine 1-phosphate signaling axis mediates neuropeptide S-induced invasive phenotype of endometriotic cells.

Endometriosis is a chronic gynecological syndrome characterized by endometrial cell invasion of the extra-uterine milieu, pelvic pain and infertility. Treatment relies on either symptomatic drugs or hormonal therapies, even though the mechanism involved in the onset of endometriosis is yet to be elucidated. The signaling of sphingolipid sphingosine 1-phosphate (S1P) is profoundly dysregulated in endometriosis. Indeed, sphingosine kinase (SK)1, one of the two isoenzymes responsible for S1P biosynthesis, and S1P1, S1P3 and S1P5, three of its five specific receptors, are more highly expressed in endometriotic lesions compared to healthy endometrium. Recently, missense coding variants of the gene encoding the receptor 1 for neuropeptide S (NPS) have been robustly associated with endometriosis in humans. This study aimed to characterize the biological effect of NPS in endometriotic epithelial cells and the possible involvement of the S1P signaling axis in its action. NPS was found to potently induce cell invasion and actin cytoskeletal remodeling. Of note, the NPS-induced invasive phenotype was dependent on SK1 and SK2 as well as on S1P1 and S1P3, given that the biological action of the neuropeptide was fully prevented when one of the two biosynthetic enzymes or one of the two selective receptors was inhibited or silenced. Furthermore, the RhoA/Rho kinase pathway, downstream to S1P receptor signaling, was found to be critically implicated in invasion and cytoskeletal remodeling elicited by NPS. These findings provide new information to the understanding of the molecular mechanisms implicated in endometriosis pathogenesis, establishing the rationale for non-hormonal therapeutic targets for its treatment.

Integrated multi-omics profiling to dissect the spatiotemporal evolution of metastatic hepatocellular carcinoma.

556 Background: Comprehensive molecular analyses of metastatic hepatocellular carcinoma (HCC) are lacking. Methods: Here, we generated genomic, transcriptomic, and T cell repertoire data coupled with digital pathology and digital spatial profiling of 257 primary, 28 relapsed, and 176 metastatic regions from 182 HCC patients. Results: Primary tumors rich in hypoxia signatures tended to give rise to polyclonal dissemination, which was associated with a poor clinical outcome. Genomic divergence between primary and metastatic HCC was high, and early dissemination was prevalent. The remarkable neoantigen intratumor heterogeneity observed in metastases was associated with decreased T cell reactivity, which may have resulted from disruptions to neoantigen presentation. We identified macroevolutionary somatic copy number alterations as highly selected events driving metastasis. Furthermore, we found that subclonal Wnt pathway mutations were not selected during spreading, whereas subclones without Wnt mutations showed a strong selective advantage for metastasis and were characterized by a reactive microenvironment rich in activated fibroblasts favoring an invasive tumor cell phenotype. Finally, metastases without Wnt mutations exhibited higher enrichment of immunosuppressive B cells that mediated terminal exhaustion of CD8+ T cells via HLA-E – NKG2A checkpoint axis. Conclusions: Collectively, our results provide a multi-dimensional dissection of the complex evolutionary process of metastasis.

Characterization of the genome and cell invasive phenotype of Vibrio diabolicus Cg5 isolated from mass mortality of Pacific oyster, Crassostrea gigas

Vibrio is an important group of aquatic animal pathogens, which has been identified as the main pathogenic factor causing mass summer mortality of Crassostrea gigas in northern China. This study aims to investigate the potential pathogenic mechanisms of Vibrio Cg5 isolate in C. gigas. We sequenced and annotated the genome of Vibrio Cg5 to analyze potential virulence factors. The gentamicin protection assays were performed with C. gigas primary cells to reveal the cell-invasive behavior of Cg5. The genome analysis showed that Cg5 was a strain of human disease-associated pathogen with multiple antibiotic resistance, and four virulence factors associated with intracellular survival were present in the genome. The gentamicin protection assays showed that Cg5 could potentially invade the cells of C. gigas, indicating that Cg5 could be a facultative intracellular pathogen of C. gigas. These results provide insights into the pathogenic mechanism of V. diabolicus, an emerging pathogenic Vibrio on aquatic animals, which would be valuable in preventing and controlling diseases in oysters.

Dihydroartemisinin inhibits liver cancer cell migration and invasion by reducing ATP synthase production through CaMKK2/NCLX.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and mitochondrial sodium/calcium exchanger protein (NCLX) are key regulatory factors in calcium homeostasis. Finding natural drugs that target regulators of calcium homeostasis is critical. Dihydroartemisinin (DHA) is considered to have anticancer effects. The present study aimed to investigate the mechanism of DHA in regulating liver cancer migration and invasion. The present study used HepG2 and HuH-7 cells and overexpressed CaMKK2 and knocked down CaMKK2 and NCLX. The antiproliferative activity of DHA on liver cancer cells was assessed through colony formation and EdU assays. Cell apoptosis was detected through YO-PRO-1/PI staining. The levels of reactive oxygen species (ROS) were measured using a ROS detection kit (DCFH-DA fluorescent probe). Cell migratory and invasive abilities were examined using wound healing and Transwell assays. The ATP production of liver cancer cells was detected using ATP fluorescent probes. Cell microfilaments were monitored for changes using Actin-Tracker Green-488. The effects of DHA on the expression of CaMKK2, NCLX, sodium/potassium-transporting ATPase subunit α-1 (ATP1A1) and ATP synthase subunit d, mitochondrial (ATP5H) were determined by western blotting and reverse transcription-quantitative PCR. The results revealed that DHA significantly inhibited proliferation, reduced ROS levels and promoted apoptosis in liver cancer cells. CaMKK2 overexpression significantly enhanced the invasive and migratory ability of liver cancer cells, whereas DHA inhibited the pro-migratory effects of CaMKK2 overexpression. DHA significantly reduced the mitochondrial ATP production and altered the arrangement of microfilaments in liver cancer cells. In addition, DHA significantly decreased the expression of CaMKK2, NCLX, ATP1A1 and ATP5H. Furthermore, by knockdown experiments of NCLX the results demonstrated that CaMKK2 downregulated the expression of ATP1A1 and ATP5H in liver cancer cells through NCLX. In conclusion, DHA may reduce ATP synthase production via the CaMKK2/NCLX signaling pathway to inhibit the invasive phenotype of liver cancer cells. It is essential to further investigate the effectiveness of DHA in the anticancer mechanism of liver cancer cells.

Centrosome amplification promotes cell invasion via cell-cell contact disruption and Rap-1 activation.

Centrosome amplification (CA) is a prominent feature of human cancers linked to tumourigenesis in vivo. Here, we report mechanistic contributions of CA induction alone to tumour architecture and extracellular matrix (ECM) remodelling. CA induction in non-tumourigenic breast cells MCF10A causes cell migration and invasion, with underlying disruption of epithelial cell-cell junction integrity and dysregulation of expression and subcellular localisation of cell junction proteins. CA also elevates expression of integrin β-3, binding partner fibronectin-1, matrix metalloprotease enzymes promoting cell-ECM attachment, ECM degradation, and a migratory and invasive cell phenotype. Using a Chicken Embryo xenograft model for in vivo validation, we show that +CA MCF10A cells invade into the chick mesodermal layer, with inflammatory cell infiltration and marked focal reactions between chorioallantoic membrane and cell graft. We also demonstrate a key role of small GTPase Rap-1 signalling through inhibition using GGTI-298, which blocked various CA-induced effects. These insights reveal that in normal cells, CA induction alone (without additional pro-tumorigenic alterations) is sufficient to confer early pro-tumourigenic changes within days, acting through Rap-1-dependent signalling to alter cell-cell contacts and ECM disruption.

An Increase in HSF1 Expression Directs Human Mammary Epithelial Cells toward a Mesenchymal Phenotype.

HSF1 is a well-known heat shock protein expression regulator in response to stress. It also regulates processes important for growth, development or tumorigenesis. We studied the HSF1 influence on the phenotype of non-tumorigenic human mammary epithelial (MCF10A and MCF12A) and several triple-negative breast cancer cell lines. MCF10A and MCF12A differ in terms of HSF1 levels, morphology, growth in Matrigel, expression of epithelial (CDH1) and mesenchymal (VIM) markers (MCF10A are epithelial cells; MCF12A resemble mesenchymal cells). HSF1 down-regulation led to a reduced proliferation rate and spheroid formation in Matrigel by MCF10A cells. However, it did not affect MCF12A proliferation but led to CDH1 up-regulation and the formation of better organized spheroids. HSF1 overexpression in MCF10A resulted in reduced CDH1 and increased VIM expression and the acquisition of elongated fibroblast-like morphology. The above-mentioned results suggest that elevated levels of HSF1 may direct mammary epithelial cells toward a mesenchymal phenotype, while a lowering of HSF1 could reverse the mesenchymal phenotype to an epithelial one. Therefore, HSF1 may be involved in the remodeling of mammary gland architecture over the female lifetime. Moreover, HSF1 levels positively correlated with the invasive phenotype of triple-negative breast cancer cells, and their growth was inhibited by the HSF1 inhibitor DTHIB.