Extracellular Matrix Detachment Research Articles

Targeting TUBB3 Suppresses Anoikis Resistance and Bone Metastasis in Prostate Cancer.

Bone metastases occur in more than 70% of advanced prostate cancer (PCa) patients, leading to a poor prognosis. Resistance to detachment-induced apoptosis, also known as anoikis, plays a crucial role in the onset of tumor metastasis. Targeting anoikis resistance is of immense therapeutic significance in repression of metastatic spread. In this study, based on an anoikis-related prognostic risk model of PCa, we identify TUBB3 as a key anoikis-related prognostic gene that is highly expressed in bone metastatic PCa. TUBB3 expression is increased in anoikis-resistant PCa cells, and TUBB3 depletion significantly reverses anoikis resistance during extracellular matrix (ECM) detachment and inhibits anoikis-resistance-induced PCa cell invasion and migration as well as epithelial-mesenchymal transition (EMT) process. TUBB3 knockdown significantly reduces αvβ3/FAK/Src axis activation, blocking its downstream oncogenic signaling. In addition, we develop bone-targeting lipid nanoparticles (BT-LNP) based on bisphosphonate-modified ionizable lipid for systemic delivery of siRNA targeting TUBB3 (siTUBB3). BT-LNP-delivered siTUBB3 therapy with localization in the bone microenvironment significantly attenuate PCa bone metastasis progression in vivo upon intravenous administration. These findings pinpoint that TUBB3, as a key regulator of anoikis resistance, is an effective therapeutic target in bone metastatic PCa and that BT-LNP-mediated systemic delivery of siTUBB3 can be developed as a novel therapeutic strategy for this disease. This article is protected by copyright. All rights reserved.

The clustering status of detached gastric cancer cells inhibits anoikis-induced ferroptosis to promote metastatic colonization

Background and purposeFerroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation. Its role in cancer metastasis remains unclear. In this study, we aimed to investigate the potential involvement of ferroptosis in gastric cancer (GC) metastasis.MethodsGC cells (AGS, MKN45, HGC27) were used to explore the role of ferroptosis in single and clustered cells with extracellular matrix (ECM) detachment in vitro. We overexpressed glutathione peroxidase 4 (GPX4) to inhibit ferroptosis and assessed the changes in cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Then tumor tissues from 54 GC patients with and without lymphatic metastasis were collected for immunohistochemical staining to investigate the expression of ferroptosis and EMT markers. Finally, Kaplan–Meier survival curves were used to investigate the relationship between overall survival and expression of GPX4 in 178 GC patients.ResultsDetached single cells had lower viability than adherent cells, but cell clustering improved their survival under matrix-detached conditions. Detached single cells exhibited an induction of iron-dependent reactive oxygen species (ROS) accumulation, glutathione (GSH) depletion, lipid peroxidation, upregulation of ACSL4, TFRC and HO-1, increased iron levels, and changes in mitochondrial morphology. Opposite effects were observed in detached clustered cells, including the upregulation of the ferroptosis suppressors GPX4 and SLC7A11. Overexpression of GPX4 inhibited ferroptosis and promoted GC cell proliferation, migration, invasion, and EMT. Immunohistochemical analysis of tumor tissues from GC patients indicated that lymphatic metastasis was associated with higher potential for ferroptosis inhibition and EMT induction. Finally, Kaplan–Meier survival curves demonstrated a significant decrease in overall survival among GC patients with high GPX4 expression.ConclusionsOur study provides the first evidence that inhibition of ferroptosis is a crucial mechanism promoting GC metastasis. GPX4 may be a valuable prognostic factor for GC patients. These findings suggest that targeting ferroptosis inhibition may be a promising strategy for GC patients with metastatic potential.Trial registration The ethical approval code of this study in Institutional Review Board of Peking Union Medical College Hospital is No: K1447.

Reprogramming of iron metabolism confers ferroptosis resistance in ECM-detached cells

Cancer cells often acquire resistance to cell death programs induced by loss of integrin-mediated attachment to extracellular matrix (ECM). Given that adaptation to ECM-detached conditions can facilitate tumor progression and metastasis, there is significant interest in effective elimination of ECM-detached cancer cells. Here, we find that ECM-detached cells are remarkably resistant to the induction of ferroptosis. Although alterations in membrane lipid content are observed during ECM detachment, it is instead fundamental changes in iron metabolism that underlie resistance of ECM-detached cells to ferroptosis. More specifically, our data demonstrate that levels of free iron are low during ECM detachment because of changes in both iron uptake and iron storage. In addition, we establish that lowering the levels of ferritin sensitizes ECM-detached cells to death by ferroptosis. Taken together, our data suggest that therapeutics designed to kill cancer cells by ferroptosis may be hindered by lack of efficacy toward ECM-detached cells.

Abstract 1381: Reprogramming of iron metabolism confers ferroptosis resistance in ECM-detached cells

Abstract Cancer cells often acquire resistance to cell death programs induced by loss of integrin-mediated attachment to extracellular matrix (ECM). Given that adaptation to ECM-detached conditions can facilitate tumor progression and metastasis, there is significant interest in effective elimination of ECM-detached cancer cells. Ferroptosis is an iron dependent non-apoptotic cell death due to catastrophic accumulation of lipid peroxidation. While the mechanism that determines ferroptosis sensitivity has been extensively investigated in attached cells, how cells respond to ferroptosis under ECM detachment is largely unknown. Here, we find that ECM-detached cells are remarkably resistant to the induction of ferroptosis. While alterations in membrane lipid content are observed during ECM-detachment, it is instead fundamental changes in iron metabolism that underlie resistance of ECM-detached cells to ferroptosis. More specifically, our data demonstrate that levels of free iron are low during ECM-detachment due to changes in both iron uptake and iron storage. In addition, we establish that lowering the levels of iron storage proteins sensitizes ECM-detached cells to death by ferroptosis. Taken together, our data suggest that therapeutics designed to kill cancer cells by ferroptosis may be hindered by lack of efficacy towards ECM-detached cells. Citation Format: Jianping He, Abigail M. Abikoye, Brett P. McLaughlin, Ryan S. Middleton, Ryan Sheldon, Russell G. Jones, Zachary T. Schafer. Reprogramming of iron metabolism confers ferroptosis resistance in ECM-detached cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1381.

P4HA1 activates HMGCS1 to promote nasopharyngeal carcinoma ferroptosis resistance and progression

Ferroptosis is a novel type of iron-dependent regulatory cell death. To date, the regulatory mechanism of ferroptosis in nasopharyngeal carcinoma (NPC) remains poorly understood. In this study, we found that the prolyl 4-hydroxylase (P4H) subunit P4HA1 protects NPC cells from erastin-induced ferroptosis by activating HMGCS1, a key enzyme in the mevalonate pathway. We also found that the P4HA1/HMGCS1 axis promoted NPC cell proliferation in vitro. In vivo, downregulation of the P4HA1/HMGCS1 axis inhibited the growth of NPC cell xenografts and enhanced the inhibitory effect of erastin on tumor growth. Extracellular matrix (ECM) detachment is an important trigger for ferroptosis. We found that the P4HA1/HMGCS1 axis promoted the ferroptosis resistance and survival of ECM-detached NPC cells. In vivo, downregulation of the P4HA1/HMGCS1 axis inhibited the lung colonization of NPC cells and enhanced the inhibitory effect of erastin on NPC lung metastasis. Moreover, the high expression of P4HA1 predicted a poor prognosis and served as a potential independent prognostic factor in patients with NPC. In conclusion, P4HA1 is a novel molecular marker of NPC ferroptosis resistance and a poor prognosis, and the P4HA1/HMGCS1 axis provides a new target for the treatment of NPC progression.

Assessment of Metabolic Pathways and Parameters in Extracellular Matrix-Detached Cells.

Changes in metabolism can alter a variety of distinct cellular parameters in a number of physiological and pathological contexts. Relatedly, the loss of integrin-mediated attachment to extracellular matrix (ECM) is now appreciated to alter metabolism in a variety of distinct fashions. As such, assays to quantify and assess metabolism during ECM detachment are critical to better understanding the cellular and molecular changes that impact the behavior and survival of ECM-detached cells. Here, we discuss assays and approaches commonly used to study metabolism during ECM detachment.

Characterization of the pleural microenvironment niche and cancer transition using single-cell RNA sequencing in EGFR-mutated lung cancer.

Background: Lung cancer is associated with a high mortality rate and often complicated with malignant pleural effusion (MPE), which has a very poor clinical outcome with a short life expectancy. However, our understanding of cell-specific mechanisms underlying the pathobiology of pleural metastasis remains incomplete. Methods: We analyzed single-cell transcriptomes of cells in pleural effusion collected from patients with lung cancer and congestive heart failure (as a control), respectively. Soluble and complement factors were measured using a multiplex cytokine bead assay. The role of ferroptosis was evaluated by GPX4 small interfering RNA (siRNA) transfection and overexpression. Results: We found that the mesothelial-mesenchymal transition (MesoMT) of the pleural mesothelial cells contributed to pleural metastasis, which was validated by lung cancer/mesothelial cell co-culture experiments. The ferroptosis resistance that protected cancer from death which was secondary to extracellular matrix detachment was critical for pleural metastasis. We found a universal presence of immune-suppressive lipid-associated tumor-associated macrophages (LA-TAMs) with complement cascade alteration in the MPE of the lung cancer patients. Specifically, upregulated complement factors were also found in the MPE, and C5 was associated with poor overall survival in the lung cancer patients with epidermal growth factor receptor mutation. Plasmacytoid dendritic cells (pDCs) exhibited a dysfunctional phenotype and pro-tumorigenic feature in the primary cancer. High expression of the gene set extracted from pDCs was associated with a poor prognosis in the lung cancer patients. Receptor-ligand interaction analysis revealed that the pleural metastatic niche was aggravated by cross-talk between mesothelial cells-cancer cells/immune cells via TNC and ICAM1. Conclusions: Taken together, our results highlight cell-specific mechanisms involved in the pathobiological development of pleural metastasis in lung cancer. These results provide a large-scale and high-dimensional characterization of the pleural microenvironment and offer a useful resource for the future development of therapeutic drugs in lung cancer.

1α,25-dihydroxyvitamin D reduction of MCF10A-ras cell viability in extracellular matrix detached conditions is dependent on regulation of pyruvate carboxylase

An emerging hallmark of cancer is cellular metabolic reprogramming to adapt to varying cellular environments. Throughout the process of metastasis cancer cells gain anchorage independence which confers survival characteristics when detached from the extracellular matrix (ECM). Previous work demonstrates that the bioactive metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25[OH]2D), suppresses cancer progression, potentially by suppressing the ability of cells to metabolically adapt to varying cellular environments such as ECM detachment. The purpose of the present study was to determine the mechanistic bases of the effects of 1,25(OH)2D on cell survival in ECM-detached conditions. Pretreatment of MCF10A-ras breast cancer cells for 3 d with 1,25(OH)2D reduced the viability of cells in subsequent detached conditions by 11%. Enrichment of 13C5-glutamine was reduced in glutamate (21%), malate (30%), and aspartate (23%) in detached compared to attached MCF10A-ras cells. Pretreatment with 1,25(OH)2D further reduced glutamine flux into downstream metabolites glutamate (5%), malate (6%), and aspartate (10%) compared to detached vehicle treated cells. Compared to attached cells, detachment increased pyruvate carboxylase (PC) mRNA abundance and protein expression by 95% and 190%, respectively. Consistent with these results, 13C6-glucose derived M+3 labelling was shown to preferentially replenish malate and aspartate, but not citrate pools, demonstrating increased PC activity in detached cells. In contrast, 1,25(OH)2D pretreatment of detached cells reduced PC mRNA abundance and protein expression by 63% and 56%, respectively, and reduced PC activity as determined by decreased 13C6-glucose derived M+3 labeling in citrate (8%) and aspartate (50%) pools, relative to vehicle-treated detached cells. While depletion of PC with doxycycline-inducible shRNA reduced detached cell viability, PC knockdown in combination with 1,25(OH)2D treatment did not additionally affect the viability of detached cells. Further, PC overexpression improved detached cell viability, and inhibited the effect of 1,25(OH)2D on detached cell survival, suggesting that 1,25(OH)2D mediates its effects in detachment through regulation of PC expression. These results suggest that inhibition of PC by 1,25(OH)2D suppresses cancer cell anchorage independence.

Integrins Cooperate With the EGFR/Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis.

Adhesion to the extracellular matrix (ECM) is required for normal epithelial cell survival. Disruption of this interaction leads to a specific type of apoptosis known as anoikis. Yet, there are physiological and pathological situations in which cells not connected to the ECM are protected from anoikis, such as during cell migration or metastasis. The main receptors transmitting signals from the ECM are members of the integrin family. However, although integrin-mediated cell-ECM anchorage has been long recognized as crucial for epithelial cell survival, the in vivo significance of this interaction remains to be weighed. In this work, we have used the Drosophila wing imaginal disc epithelium to analyze the importance of integrins as survival factors during epithelia morphogenesis. We show that reducing integrin expression in the wing disc induces caspase-dependent cell death and basal extrusion of the dead cells. In this case, anoikis is mediated by the activation of the JNK pathway, which in turn triggers expression of the proapoptotic protein Hid. In addition, our results strongly suggest that, during wing disc morphogenesis, the EGFR pathway protects cells undergoing cell shape changes upon ECM detachment from anoikis. Furthermore, we show that oncogenic activation of the EGFR/Ras pathway in integrin mutant cells rescues them from apoptosis while promoting their extrusion from the epithelium. Altogether, our results support the idea that integrins promote cell survival during normal tissue morphogenesis and prevent the extrusion of transformed cells.

CEMIP promotes extracellular matrix‐detached prostate cancer cell survival by inhibiting ferroptosis

Cells detached from the extracellular matrix (ECM) can trigger different modes of cell death, and the survival of ECM‐detached cells is one of the prerequisites for the metastatic cascade. Ferroptosis, a form of iron‐dependent programmed cell death, has recently been found to be involved in matrix‐detached cancer cells. However, the molecular mechanisms by which ECM‐detached cells escape ferroptosis are not fully understood. Here, we observed that cell migration‐inducing protein (CEMIP) upregulation facilitates ferroptosis resistance during ECM detachment by promoting cystine uptake in prostate cancer (PCa) cells. Meanwhile, silencing CEMIP causes it to lose its ability to promote cystine uptake and inhibit ferroptosis. Mechanistically, the interaction of CEMIP with inositol 1,4,5‐trisphosphate receptor type 3 (ITPR3) modulates calcium ion (Ca2+) leakage from the endoplasmic reticulum, activating calcium/calmodulin‐dependent protein kinase II (CaMKII), which further facilitates nuclear factor erythroid 2‐related factor 2 (NRF2) phosphorylation and nuclear localization, leading to elevated transcription of solute carrier family 7 member 11 (SLC7A11), a glutamate/cystine antiporter, in PCa cells. Our findings delineate a novel role of CEMIP in ferroptosis resistance during ECM detachment and provide new insights into therapeutic strategies for metastatic PCa.

Untargeted Metabolomics Showed Accumulation of One-Carbon Metabolites to Facilitate DNA Methylation during Extracellular Matrix Detachment of Cancer Cells.

Tumor cells detached from the extracellular matrix (ECM) undergo anoikis resistance and metabolic reprogramming to facilitate cancer cell survival and promote metastasis. During ECM detachment, cancer cells utilize genomic methylation to regulate transcriptional events. One-carbon (1C) metabolism is a well-known contributor of SAM, a global substrate for methylation reactions, especially DNA methylation. DNA methylation-mediated repression of NK cell ligands MICA and MICB during ECM detachment has been overlooked. In the current work, we quantitated the impact of ECM detachment on one-carbon metabolites, expression of 1C regulatory pathway genes, and total methylation levels. Our results showed that ECM detachment promotes the accumulation of one-carbon metabolites and induces regulatory pathway genes and total DNA methylation. Furthermore, we measured the expression of well-known targets of DNA methylation in NK cell ligands in cancer cells, namely, MICA/B, during ECM detachment and observed low expression compared to ECM-attached cancer cells. Finally, we treated the ECM-detached cancer cells with vitamin C (a global methylation inhibitor) and observed a reduction in the promoter methylation of NK cell ligands, resulting in MICA/B re-expression. Treatment with vitamin C was also found to reduce global DNA methylation levels in ECM-detached cancer cells.

The Histone H3K27me3 Demethylases KDM6A/B Resist Anoikis and Transcriptionally Regulate Stemness-Related Genes.

Epithelial cancer cells that lose attachment from the extracellular matrix (ECM) to seed in a distant organ often undergo anoikis’s specialized form of apoptosis. Recently, KDM3A (H3K9 demethylase) has been identified as a critical effector of anoikis in cancer cells. However, whether other histone demethylases are involved in promoting or resisting anoikis remains elusive. We screened the major histone demethylases and found that both H3K27 histone demethylases, namely, KDM6A/B were highly expressed during ECM detachment. Inhibition of the KDM6A/B activity by using a specific inhibitor results in reduced sphere formation capacity and increased apoptosis. Knockout of KDM6B leads to the loss of stem cell properties in solitary cells. Furthermore, we found that KDM6B maintains stemness by transcriptionally regulating the expression of stemness genes SOX2, SOX9, and CD44 in detached cells. KDM6B occupies the promoter region of both SOX2 and CD44 to regulate their expression epigenetically. We also noticed an increased occupancy of the HIF1α promoter by KDM6B, suggesting its regulatory role in maintaining hypoxia in detached cancer cells. This observation was further strengthened as we found a significant positive association in the expression of both KDM6B and HIF1α in various cancer types. Overall, our results reveal a novel transcriptional program that regulates resistance against anoikis and maintains stemness-like properties.

ATF4/CEMIP/PKC\u03b1 promotes anoikis resistance by enhancing protective autophagy in prostate cancer cells

The survival of cancer cells after detaching from the extracellular matrix (ECM) is essential for the metastatic cascade. The programmed cell death after detachment is known as anoikis, acting as a metastasis barrier. However, the most aggressive cancer cells escape anoikis and other cell death patterns to initiate the metastatic cascade. This study revealed the role of cell migration-inducing protein (CEMIP) in autophagy modulation and anoikis resistance during ECM detachment. CEMIP amplification during ECM detachment resulted in protective autophagy induction via a mechanism dependent on the dissociation of the B-cell lymphoma-2 (Bcl-2)/Beclin1 complex. Additional investigation revealed that acting transcription factor 4 (ATF4) triggered CEMIP transcription and enhanced protein kinase C alpha (PKCα) membrane translocation, which regulated the serine70 phosphorylation of Bcl-2, while the subsequent dissociation of the Bcl-2/Beclin1 complex led to autophagy. Therefore, CEMIP antagonization attenuated metastasis formation in vivo. In conclusion, inhibiting CEMIP-mediated protective autophagy may provide a therapeutic strategy for metastatic prostate cancer (PCa). This study delineates a novel role of CEMIP in anoikis resistance and provides new insight into seeking therapeutic strategies for metastatic PCa.

Abstract 2873: Comparison of metastatic and nonmetastatic breast cancer cell survival in glutamine depleted extracellular matrix detached conditions

Abstract For women, breast cancer is the most diagnosed cancer, with metastasis being the primary cause of most breast cancer-related deaths. During metastasis, cells detach from the extracellular matrix (ECM) to move to a secondary site. Unlike normal cells, metastatic cells are resistant to cell death upon ECM detachment. The amino acid glutamine is used as an energy source during metastatic progression via replenishing the TCA cycle. Breast cancer cells have decreased viability in the absence of glutamine, but the effect of glutamine in ECM detached conditions in metastatic cells is not known. We hypothesize that metastatic and non-metastatic cells have a decreased viability in ECM detachment in glutamine deprived conditions. In this study, metastatic MCF10CA1a cells and non-metastatic Harvey-ras oncogene transfected MCF10A-ras cells were employed. As expected, in ECM detached conditions, the MCF10A-ras cells had a greater decrease in viability (58.4%) in comparison to the MCF10CA1a cells (32.7%). However, in glutamine deprived conditions, the MCF10CA1a cells decrease in viability was 15.2% greater compared to the MCF10A-ras cells. mRNA expression of the major glutamine transporter, SLC1A5, was decreased in detached conditions in both cell lines, but to a greater extent in the MCF10A-ras cells (70.04%) compared to the MCF10CA1a cells (20.15%). Additionally, mRNA expression in detached conditions of glutamate dehydrogenase, the enzyme that metabolizes the downstream glutamine metabolite glutamate to α-ketoglutarate (αKG), was decreased by 77% in the non-metastatic cells, but increased by 23% in the metastatic cells. Additionally, mRNA expression of GOT1, a gene which encodes for the transaminase enzyme which synthesizes αKG and aspartate from glutamate and oxaloacetate, decreased 73% in detached conditions in the non-metastatic cells but was unchanged in the metastatic cells. Moreover, following detachment the glutamine synthesizing enzyme, glutamine synthetase mRNA expression was increased by 81.92% in the non-metastatic cells and 88.43% in the metastatic cells. In summary, the greater effect of glutamine depletion on viability, and the maintained expression of glutamine metabolizing enzymes in the metastatic cells suggest greater dependence on glutamine metabolism in ECM detached conditions compared to non-metastatic cells. Comparison of metastatic and non-metastatic breast cancer cells will provide insights to direct the development of new targets for the prevention of breast cancer metastasis. Citation Format: Kanika K. Garg, Madeline Sheeley, Dorothy Teegarden. Comparison of metastatic and nonmetastatic breast cancer cell survival in glutamine depleted extracellular matrix detached conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2873.

Abstract 2327: Pyruvate carboxylase rescues MCF10A-ras cell viability in extracellular matrix detached conditions

Abstract Breast cancer is the most diagnosed cancer in women, and metastasis accounts for 90% of cancer related deaths. In order to metastasize, cancer cells must survive in conditions where they are detached from the extracellular matrix (ECM). Untransformed cells decrease energy metabolism when detached from ECM, but oncogenic transformation results in rescued energy metabolism of detached cells. Expression of the anaplerotic enzyme pyruvate carboxylase (PC) is required for breast to lung metastasis, and may play a role in promoting ECM detached cell survival by supporting metabolic plasticity. PC activity results in the carboxylation of pyruvate to synthesize the TCA cycle intermediate oxaloacetate (OAA). We hypothesize that PC expression is required for cell survival in ECM detached conditions. The Harvey-ras transformed MCF10A human breast epithelial cell line (MCF10A-ras) were utilized for these studies. ECM detachment was modeled using Poly-HEMA coated plates in combination with MTT assays to measure cell viability. qPCR was used to quantify mRNA abundance of metabolic genes. Protein expression was quantified using western blot. PC activity was determined using [13C6]-glucose flux analysis by comparing 3 carbon labeled (M+3) citrate, malate, and aspartate. Results show a 94% increase in PC mRNA expression and a 90% increase in PC protein expression, in detached cells compared to attached cells. In addition PC knockdown decreases viability in ECM detached conditions significantly by 15%, while PC overexpression increases viability by 17%. Further, replenishing the TCA cycle with OAA, the product of the PC reaction, rescues PC knockdown cells by 14%. Increased PC activity in detached cells was determined by a 23% increase in M+3 malate and a 61% increase in M+3 aspartate. However, an 11% decrease in M+3 citrate was observed, suggesting that detached cells do not utilize increased PC expression to replenish the TCA cycle intermediate, citrate. Given the larger relative increase in PC activity demonstrated by M+3 aspartate, we hypothesized that PC activity is required for detached cell survival to provide substrate for aspartate synthesis. Providing aspartate (2.5 mM), a downstream product of PC, in detached cells with PC knockdown rescued cell viability with no benefit of aspartate supplementation in detached PC expressing cells. Further, the aspartate synthesizing enzyme, glutamate OAA transferase 2 (GOT2) mRNA expression was unchanged in detached cells. In summary PC expression and activity is increased in detached cells compared to attached cells, and OAA produced by the PC reaction is utilized by cells to synthesize aspartate and maintain cell viability in detached conditions. PC may be a useful target for future treatments aimed at preventing metastasis. Citation Format: Madeline P. Sheeley, Dorothy Teegarden. Pyruvate carboxylase rescues MCF10A-ras cell viability in extracellular matrix detached conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2327.

Abstract 1980: Non-canonical regulation and function of anti-apoptotic protein c-FLIP

Abstract The metastasis of cancer cells to distant sites accounts for over 90% of cancer related deaths. In order to metastasize, cancer cells must be able to survive during extracellular matrix (ECM) detachment, namely by blocking anoikis (a caspase-dependent cell death) and restoring metabolic deficiencies. Such barriers during tumorigenesis are abrogated through activation of oncogenic signaling, and/or inhibition of tumor suppressors. Elucidating the signaling pathways involved in cancer cell metastasis is a crucial step in designing effective therapeutic interventions. Surprisingly, our investigation has revealed that cellular FLICE-like Inhibitory Protein (c-FLIP), a protein well known for its pro-survival function, instead antagonizes the viability of ECM-detached cancer cells. Additionally, we demonstrated that signaling emanating from oncogenic growth factor receptors results in the downregulation of c-FLIP expression providing additional evidence that cancer cells may benefit from diminished FLIP levels. Interestingly, this repression of c-FLIP expression by activated oncogenes is a consequence of PI3K signaling. Our study also suggests that low c-FLIP levels are beneficial in cancer cells due to capacity of c-FLIP to induce β-catenin degradation. Taken together, our study has revealed an unexpected role for c-FLIP in restricting the viability of tumor cells and raises the idea that the c-FLIP may have context-dependent pro- and anti-cell death roles during tumorigenesis. Citation Format: Matyas Abel Tsegaye, Kyle McGeehan, Mati Nemera, Zachary T. Schafer. Non-canonical regulation and function of anti-apoptotic protein c-FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1980.

Mechanisms for Modulating Anoikis Resistance in Cancer and the Relevance of Metabolic Reprogramming.

The attachment of cells to the extracellular matrix (ECM) is the hallmark of structure–function stability and well-being. ECM detachment in localized tumors precedes abnormal dissemination of tumor cells culminating in metastasis. Programmed cell death (PCD) is activated during tumorigenesis to clear off ECM-detached cells through “anoikis.” However, cancer cells develop several mechanisms for abrogating anoikis, thus promoting their invasiveness and metastasis. Specific factors, such as growth proteins, pH, transcriptional signaling pathways, and oxidative stress, have been reported as drivers of anoikis resistance, thus enhancing cancer proliferation and metastasis. Recent studies highlighted the key contributions of metabolic pathways, enabling the cells to bypass anoikis. Therefore, understanding the mechanisms driving anoikis resistance could help to counteract tumor progression and prevent metastasis. This review elucidates the dynamics employed by cancer cells to impede anoikis, thus promoting proliferation, invasion, and metastasis. In addition, the authors have discussed other metabolic intermediates (especially amino acids and nucleotides) that are less explored, which could be crucial for anoikis resistance and metastasis.

SGK1 signaling promotes glucose metabolism and survival in extracellular matrix detached cells

Loss of integrin-mediated attachment to extracellular matrix (ECM) proteins can trigger a variety of cellular changes that affect cell viability. Foremost among these is the activation of anoikis, caspase-mediated cell death induced by ECM detachment. In addition, loss of ECM attachment causes profound alterations in cellular metabolism, which can lead to anoikis-independent cell death. Here, we describe a surprising role for serum and glucocorticoid kinase-1 (SGK1) in the promotion of energy production when cells are detached. Our data demonstrate that SGK1 activation is necessary and sufficient for ATP generation during ECM detachment and anchorage-independent growth. More specifically, SGK1 promotes a substantial elevation in glucose uptake because of elevated GLUT1 transcription. In addition, carbon flux into the pentose phosphate pathway (PPP) is necessary to accommodate elevated glucose uptake and PPP-mediated glyceraldehyde-3-phosphate (G3P) is necessary for ATP production. Thus, our datashow SGK1 as master regulator of glucose metabolism and cell survival during ECM-detached conditions.

Compound C, a Broad Kinase Inhibitor Alters Metabolic Fingerprinting of Extra Cellular Matrix Detached Cancer Cells.

Most of the cancer related deaths are caused mainly by metastasis. Therefore, it is highly important to unfold the major mechanisms governing metastasis process in cancer. Throughout the metastatic cascade, cells need the ability to survive without attachment to neighboring cells and the original Extra Cellular Matrix (ECM). Recent reports showed that loss of ECM attachment shifts cancer cell metabolism towards glycolysis mostly through hypoxia. However, AMPK, a master metabolic regulator was also found to be upregulated under ECM detached conditions. Therefore, in this work we aimed to understand the consequences of targeting AMPK and other metabolic kinases by a broad kinase inhibitor namely Compound C in ECM detached cancer cells. Results showed that Compound C impacts glycolysis as evident by increased levels of pyruvate, but reduces its conversion to lactate thereby negatively regulating the Warburg effect. Simultaneously, Compound C induces block at multiple levels in TCA cycle as evident from accumulation of various TCA metabolites. Interestingly Compound C significantly reduces glutamine and reduced glutathione levels, suggesting loss of antioxidant potential of ECM detached cancer cells. Further, we found increased in metabolites associated with nucleotide synthesis, one carbon metabolism and PPP pathway during Compound C treatment of ECM detached cells. Finally, we also found induction in metabolites associated with DNA damage in ECM detached cancer cells during Compound C treatment, suggesting DNA damage regulatory role of metabolic kinases. Overall, our results showed that Compound C represses pyruvate to lactate conversion, reduces antioxidant potential and invokes DNA damage in ECM detached cancer cells. Our data provides a comprehensive metabolic map of ECM detached cancer cells that can be targeted with a broad kinase inhibitor, is Compound C. The data can be used for designing new combinational therapies to eradicate ECM detached cancer cells.

Molecular profiling of epigenetic landscape of cancer cells during extracellular matrix detachment

During cancer, a major challenge faced by oncologists is the treatment of metastasis; a leading cause of cancer-related deaths around the world. Metastasis involves a highly ordered sequence of events starting with the detachment of tumor cells from the extracellular matrix (E.C.M.). In normal cells, detachment from E.C.M. triggers programmed cell death, termed anoikis. However, tumor cells dodge their way to anoikis and spread to distant sites for initiating the metastatic program. In this work, we explored the impact of E.C.M. detachment on the expression of some major oncogenic histone methyltransferases. Results showed both EZH2 expression and its enzymatic activity were significantly increased in E.C.M. detached cancer cells when compared to the attached cells. Inhibition of EZH2 results in a significant reduction in cell proliferation, spheroids size, and induction in apoptosis in E.C.M. detached cells. Furthermore, we observed a reduction in EZH2 expression levels in single cells when compared to clusters of E.C.M. detached cells. Finally, we combined the EZH2 inhibition with AMPK, known to be highly expressed in E.C.M. detached cancer cells and observed antagonistic effects between the two pathways. The observed results clearly showed that E.C.M. detached cancer cells require oncogenic EZH2 and can be targeted by EZH2 inhibitors.