Cancer Cell Plasticity Research Articles

Abstract 4251: Development and characterization of an epiregulin antibody-drug conjugate for targeting colorectal cancer cell plasticity

Abstract Colorectal cancer (CRC) is the second leading cause of cancer related deaths in the United States. This is largely due to metastasis, tumor relapse, and the vast heterogeneity of CRC tumors leading to treatment failure. Cancer stem cells (CSCs) are a small population of tumor-initiating cells thought to act as immortal seeds that can form metastases and contribute to tumor relapse. CSCs have been shown to exhibit plasticity or the ability to transition between differentiated and undifferentiated states to evade treatment and to promote tumor progression. Epiregulin (EREG) is an EGFR family ligand that is highly expressed in both states, as well as in treatment resistant CRCs of various mutation statuses. We showed that knock-out of EREG in a CRC cell line resulted in significant tumor inhibition in vivo, suggesting EREG is a promising target for therapeutic development. Therefore, we generated EREG-targeted antibody-drug conjugates (ADCs) to act as guided missiles for the delivery of cytotoxic drugs into EREG-expressing tumors to overcome plasticity and resistance in CRC. To develop a novel EREG ADC, we cloned and produced three separate EREG monoclonal antibody (mAbs) and tested them for binding, the ability to internalize into CRC cells, and to neutralize EREG activity. All three EREG mAbs were shown to bind with high affinity to EREG tethered to the surface of CRC cells and internalize to the lysosome, which is important for processing the release of drug from the ADC. We then conjugated the highest affinity EREG mAb and a non-targeting isotype control antibody to a highly cytotoxic drug via a cleavable peptide linker. EREG ADCs were evaluated in vitro against a wide panel of CRC cell lines of various Kras, Braf, and PI3CKA mutational statuses and different levels of EREG expression. Drug efficacy was determined using cytotoxicity assays to measure changes in CRC cell survival with escalating doses of unconjugated control antibody and EREG mAb, as well as control ADC (cADC) and EREG ADC. EREG mAb alone did not result in significant CRC cell death. However, EREG ADCs exhibited potent cell-killing in EREG-expressing CRC cells, whereas minimal effects were observed with cADC. Future progress will involve testing the safety and efficacy of EREG ADCs against patient-derived tumor xenograft models of CRC in vivo. These preliminary results demonstrate the therapeutic potential of EREG ADCs for targeting plasticity and drug resistance in CRC. Citation Format: Joan Jacob, Zhengdong Liang, Kendra Carmon. Development and characterization of an epiregulin antibody-drug conjugate for targeting colorectal cancer cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4251.

Abstract 2995: Autophagy as a key driver of waterpipe smoke-induced lung cancer cell plasticity and resistance

Abstract Smoking is the number one risk factor for lung cancer and tobacco smokers are at 20 to 40 times higher risk of developing lung cancer in comparison to non-smokers. We have previously shown that exposing lung cancer cell lines to tobacco smoke condensate generated from waterpipe (WP) interferes with cell proliferation, cell plasticity, DNA damage and tumor cell recognition and killing by natural killer (NK) cells. Global transcriptomic analysis identified an expression profile of genes that best distinguished treated and non-treated cells involving several pathways including those involved in epithelial to mesenchymal transition (EMT) and stemness. Because of the important role of autophagy in tumor resistance and progression, we investigated its relationship with WP smoking. We first showed an activation of autophagy as reflected by LC3 and P62 in lung cancer cell lines in response to WPS. Interestingly, our data revealed that WPS-induced EMT and cancer stemness involved autophagy induction. The autophagy response in smokers with lung adenocarcinoma as compared to non-smokers with lung adenocarcinoma was investigated using TCGA lung adenocarcinoma bulk RNA-seq dataset with the available patient metadata on smoking status. Preliminary results based on a machine learning classification model using Random Forest indicate that smokers have an increase in autophagy activating genes as compared to non-smokers. Comparative analysis of lung adenocarcinoma molecular signatures in affected patients with a long-term active exposure to smoke compared to patients who have not had any active exposure to smoke indicate a higher tumor mutational burden, CD8+ T-cell level and lower dysfunction level in smokers. Multivariate regression analyses confirm these results even after controlling for confounding factors like age, gender, and tumor stage. While the checkpoint genes tested PD1, PDL1, PDL2 and CTLA-4, had no difference between smokers and non-smokers, B7-1, B7-2, IDO1 and CD200R1 had higher expression in non-smokers than smokers. Because multiple factors in the tumor microenvironment dictate the success of immunotherapy, in addition to the expression of immune checkpoint genes, our analysis explains why patients who are smokers with lung adenocarcinoma respond better to immunotherapy, even though there are no relative differences in immune checkpoint genes in the two cohorts. Therefore, targeting autophagy in lung adenocarcinoma patients in combination with checkpoint inhibitors, targeted therapies or chemotherapy should be considered in smoker patients with lung adenocarcinoma. Citation Format: Rania F. Zaarour, Mohak Sharda, Bilal Azakir, Goutham Hassan Venkatesh, Raefa Abou Khouzam, Ayesha Rifath, Zohra Nizami, Fatima Abdullah, Fatin Mohammad, Husam Nawafleh, Yehia ElSayed, Salem Chouaib. Autophagy as a key driver of waterpipe smoke-induced lung cancer cell plasticity and resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2995.

Abstract LB023: Thyroid hormone receptor signaling promotes antiandrogen resistance and bone metastasis in prostate cancer

Abstract Background: Systemic factors from the tumor microenvironment (TME) promote prostate cancer (PCa) progression, metastasis, and drug resistance. Therefore, the identification of the systemic factors that can modulate the tumor cells and promote metastases will be critical to improve the efficacy of clinical treatment. Both mechanistic and functional studies revealed that acquisition of an open chromatin state on master regulators or cis-regulatory elements allow them to respond readily to microenvironment signals, thereby increasing cancer cell plasticity, tumorigenicity and malignant potential. Accordingly, analysis of open chromatin regions can help prioritize and identify risk of functionally noncoding variants. Given the power of open chromatin profiling on environmental risk prediction, we hypothesized that analysis of open chromatin regions may enable us to recognize functional trans-regulatory elements with their relevant systemic factors. Methods and Results: In human prostate cancer cells, AR blockage by enzalutamide can induces cell reprogramming, as well as transcriptional dysregulation of the heterochromatin compaction pathway. Hence, we proposed that the cells were undergoing chromatin decondensation followed by epigenetic reprogramming. To detect the genome-wide open chromatin changes in this process, we performed time course ATAC-seq and H3K27ac ChIP-seq experiments on LNCaP cells treated with enzalutamide over a 2 weeks period. Bioinformatic analysis was performed to identify the “open” enhancers and the transcriptional factors potentially bound to these enhancers. Integrated transcription factor binding motif analysis of metastatic castration-resistant prostate cancer (mCRPC) associated enhancer regions (GSE130408) and multiplexed siRNA screening revealed Thyroid Hormone Receptor Beta (THRB) as the top candidate. THRB is regulated by the thyroid hormone (T3), a systemic nuclear hormone that, in enzalutamide pretreated LNCaP, VCaP and 3PDXs, is able to induce subsequent reprogramming to a fully enzalutamide-resistant and metastatic state. Interestingly, we observed that THRB regulated genes are upregulated in T3-induced resistant prostate cancer cells. Consistently, the THRB protein, TRβ, is abundant in several aggressive PCa cell lines. Notably, knockdown of THRB reverted the growth and the bone metastatic potential of highly aggressive PCa models, such as Ptenpc-/-Smad4pc-/- and PC3M cells. Furthermore, both transcriptomic and functional analyses identified RUNX2 as a transcriptional target of THRB. In summary, we identify a novel mechanism of THRB-mediated tumor progression and drug resistance in PCa cells. Conclusion: Epidemiologic evidence indicates a higher incidence of PCa in men with elevated thyroid hormone levels. Accordingly, our results suggest a new mechanism for thyroid hormone signaling regulating metastasis potential and drug resistance. This study will provide a new perspective to identify the signals from the TME that may increase the risk of PCa progression. Citation Format: Yan Wang, Gurrapu Harsha, Dhiraj Kumar, Josue Curto, Xiaobo Wang, Filippo Giancotti. Thyroid hormone receptor signaling promotes antiandrogen resistance and bone metastasis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB023.

Abstract 3781: Expression of ovarian cancer specific Drp1 splice variants regulate mitochondrial heterogeneity and cell plasticity during tumor progression

Abstract Mitochondrial shape is integral for its proper function and is maintained by a dynamic balance between the events of fission and fusion. Hence, a disruption in the balance is detrimental and has been associated with multiple pathologies including tumorigenesis. We noticed significant heterogeneity in mitochondrial morphology and function in ovarian cancer, which remains the deadliest gynecologic malignancy to date. We discovered that heterogenous mitochondrial dynamics in ovarian cancer cells were associated with specific transcript variant signatures of the fission protein Drp1 (encoded by the gene DNM1L), the primary GTPase responsible for mitochondrial fission. While several Drp1 splice variants have been reported, few studies have linked expression and potential interplay of splice variants of Drp1 on mitochondrial dynamics and function with pathophysiology especially in ovarian cancer. We used 3’RACE, western blotting and LC-MS/MS proteomics analysis to establish the identity of the major Drp1 splice variants expressed in ovarian cancer. We found ovarian cancer cell lines as well as patient-ascites derived cells, predominantly express two Drp1 variants: a transcript including both exons 16 and 17 (16/17) and a transcript lacking exon 16 (-/17). We also validated our findings in TCGA ovarian cancer specimens by analyzing Drp1 splice variant transcripts following annotation of TCGA raw RNAseq data and Salmon expression analysis. Our TCGA analysis of these variants highlighted significant difference in overall survival of ovarian cancer patients. Samples with high Drp1(-/17) expression were associated with poorer overall survival compared to those predominantly expressing Drp1(16/17). Furthermore, carrying out gene set enrichment analysis (GSEA) on TCGA specimens split by high expression of these two variants showed enrichment of distinct gene expression signatures. Overexpression and splice variant specific siRNA knockdown studies demonstrated that Drp1 variants have unique localization and effects on mitochondrial morphology and function. Furthermore, metabolic profiling and 13C metabolic flux analysis highlighted variant specific alterations in mitochondrial metabolic pathways and the TCA cycle. Drp1(-/17) expression enhanced mitochondrial respiratory function and as previously shown, Drp1(-/17) associated with both mitochondria and microtubules, potentially implying a more regulated fission activity as a consequence of controlled subcellular localization. Additionally, Drp1(-/17) was enriched and associated with quiescent phenotype compared to more proliferative phenotype of Drp1(16/17). Hence, expression of distinct Drp1 splice variants may be a novel mechanism to regulate mitochondrial fission, and integral to ovarian cancer cell plasticity under different selection pressures during tumor progression. Citation Format: Zaineb Javed, Dong-Hui Shin, Weihua Pan, Amal Taher Elhaw, Priscilla Tang, Rebecca Phaeton, Mohamed Trebak, Vonn Walter, Nadine Hempel. Expression of ovarian cancer specific Drp1 splice variants regulate mitochondrial heterogeneity and cell plasticity during tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3781.

Modelling the Tumor Microenvironment: Recapitulating Nano- and Micro-Scale Properties that Regulate Tumor Progression

Breast cancer remains a significant burden with 1 in 8 women affected and metastasis posing a significant challenge for patient survival. Disease progression involves remodeling of the extracellular matrix (ECM). In breast cancer, tissue stiffness increases owing to an increase in collagen production by recruited cancer-associated fibroblasts (CAFs). These stromal modifications are notable during primary tumor growth and have a dualistic action by creating a hard capsule to prevent penetration of anti-cancer therapies and forming a favorable environment for tumor progression. Remodeling of the tumor microenvironment immediately presented to cells can include changes in protein composition, concentration and structural arrangement and provides the first mechanical stimuli in the metastatic cascade. Not surprisingly, metastatic cancer cells possess the ability to mechanically adapt, and their adaptability ensures not only survival but successful invasion within altered environments. In the past decade, the importance of the microenvironment and its regulatory role in diseases have gained traction and this is evident in the shift from plastic culture to the development of novel biomaterials that mimic in vivo tissue. With these advances, elucidations can be made into how ECM remodeling and more specifically, altered cell-ECM adhesions, regulate tumor growth and cancer cell plasticity. Such enabling tools in mechanobiology will identify fundamental mechanisms in cancer progression that eventually help develop preventative and therapeutic treatment from a clinical perspective. This review will focus on current platforms engineered to mimic the micro and nano-properties of the tumor microenvironment and subsequent understanding of mechanically regulated pathways in cancer.

CREB1 contributes colorectal cancer cell plasticity by regulating lncRNA CCAT1 and NF-κB pathways.

The CREB1 gene encodes an exceptionally pleiotropic transcription factor that frequently dysregulated in human cancers. CREB1 can regulate tumor cell status of proliferation and/or migration; however, the molecular basis for this switch involvement in cell plasticity has not fully been understood yet. Here, we first show that knocking out CREB1 triggers a remarkable effect of epithelial-mesenchymal transition (EMT) and leads to the occurrence of inhibited proliferation and enhanced motility in HCT116 colorectal cancer cells. By monitoring 45 cellular signaling pathway activities, we find that multiple growth-related pathways decline significantly while inflammatory pathways including NF-κB are largely upregulated in comparing between the CREB1 wild-type and knocked out cells. Mechanistically, cells with CREB1 knocked out show downregulation of MYC as a result of impaired CREB1-dependent transcription of the oncogenic lncRNA CCAT1. Interestingly, the unbalanced competition between the coactivator CBP/p300 for CREB1 and p65 leads to the activation of the NF-κB pathway in cells with CREB1 disrupted, which induces an obvious EMT phenotype of the cancer cells. Taken together, these studies identify previously unknown mechanisms of CREB1 in CRC cell plasticity via regulating lncRNA CCAT1 and NF-κB pathways, providing a critical insight into a combined strategy for CREB1-targeted tumor therapies.

MODL-20. Metabolic rewiring support the onset of chemotherapy resistance in medulloblastoma

Abstract Medulloblastoma (MB) is the deadliest brain tumor of childhood, intrinsically characterized by fast growth, high invasiveness, and resistance to treatments. With the aim to deepen the molecular basis of MB aggressiveness and recurrence, we established an in vitro model of MB resistance to chemotherapy, in which, a weekly exposure to a cocktail of chemotherapeutics commonly used in MB treatment (Vincristine, Etoposide, Cisplatin, Cyclophosphamide – VECC) induces the selection of cells that progressively acquire resistance to subsequent VECC treatments. Preliminary data on our model of MB resistance show that resistant cells induce the activation of the two main regulator of pentose phosphate pathway TKT and G6PD via the activation of Nrf2 transcriptional activity. Moreover, resistant cells show an increase in hypoxia inducible factor-1α (HIF-1α) together with an augmented expression of glycolytic enzymes HK1, PFKB-3, PDK1 and LDHA and increased glycolytic capacity. Interestingly, enrichment analysis on label free mass spectrometry data reveal that the most significant terms deriving from the upregulated proteins, that characterized resistant cells, were related to metabolic processes such as “carbon metabolism”, “fatty acid beta oxidation”, “tricarboxylic acid cycle” and “carboxylic acid catabolic processes”.Consistently with recent studies that highlight the relevance of metabolic plasticity of cancer cells in chemotherapy adaptation, our data suggest a metabolic uncoupling in which MB resistant cells, through the alteration of peculiar metabolic processes, may satisfy their altered energetic demands through alternative metabolic pathways. In this way, the generation of a new balance of intracellular metabolites finally provide increased resistance to external insults (i.e., chemotherapeutics) and a greater ability of detoxifying the intracellular compartments.

CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

Chemotherapy is the mainstay for the treatment of non-small cell lung cancer (NSCLC). However, NSCLC cells are either intrinsically chemoresistant or rapidly develop therapy resistance. Cancer stem cells (CSCs) are widely recognized as the cell population responsible for resistance to systemic therapies, but the molecular responses of CSCs to chemotherapeutic agents are largely unknown. We identified the embryonic protein CRIPTO in stem cell-enriched spheroid cultures of adenocarcinoma (AC) and squamous cell carcinoma (SCC) derived from NSCLC surgical specimens. The CRIPTO-positive population had increased clonogenic capacity and expression of stem cell-related factors. Stemness-related properties were also obtained with forced CRIPTO expression, whereas CRIPTO downregulation resulted in cell cycle blockade and CSCs death. Cell populations positive and negative for CRIPTO expression were interconvertible, and interfering with their reciprocal equilibrium resulted in altered homeostasis of cell expansion both in spheroid cultures and in tumor xenografts. Chemotherapy treatment of NSCLC cells resulted in reduction of cell number followed by increased CRIPTO expression and selective survival of CRIPTO-positive cells. In NSCLC tumor xenografts, chemotherapeutic agents induced partial cell death and tumor stabilization followed by CRIPTO overexpression and tumor progression. Altogether, these findings indicate CRIPTO as a marker of lung CSCs possibly implicated in cancer cell plasticity and post-chemotherapy tumor progression.

Abstract B029: Epigenetic adaptability as a targetable trait underlying therapy resistance

Abstract Despite the development of effective targeted agents for the treatment of cancer, targeted therapies eventually fail in advanced, metastatic non-small cell lung cancer (NSCLC) as tumors acquire resistance. Historically, acquired therapy resistance has been attributed to genetic mutational mechanisms. Recent growing body of evidence indicates the importance of non-genetic (epigenetic) mechanisms as essential contributors to both initial ability of cells to survive therapy as well as bona fide resistance. Most commonly, resistance through epigenetic mechanisms is viewed from a perspective of well-defined specific phenotypic states, such as “epithelial mesenchymal transition” (EMT) and “stemness”. However, our resent study showed that the same parental sub-populations under selection with distinct Tyrosine kinase inhibitors (TKIs) give rise to predictably distinct TKIs-specific resistance, indicating that adaptability/phenotypic plasticity in general, rather than specific phenotypic states, might be key for ability of cells to evolve resistance to therapies. Therefore, we hypothesized that gradual, multifactorial development of resistance is fueled by cancer cell plasticity that could be exploited to discover vulnerabilities of therapy resistant cell state. Consequently, targeting of key molecular machinery responsible for this plasticity/adaptability is expected to delay acquisition of resistance. To this end, we took advantage of observations in a cell line model of ALK+ NSCLC, H3122, that develops strong, predictable resistance, when subjected to different clinically approved ALK-TKIs, primarily through non-mutational mechanisms. We found that individual single cell derived subclones display reproducible differences in the ability to develop resistance to ALK-TKIs and unrelated stressors, including distinct pharmacological inhibitors, physical stressors, growth factor deprivation. In parallel as an unbiased approach, we asked whether distinct stressors select for cells with shared clonal origin, we performed in vitro selection with different TKIs as well as other stressor and in vivo metastatic assays using H3122 cells labelled with unique barcodes by complex Clone-Tracer library followed by next generation sequencing (NGS). Next, we attempted to identify targetable molecular mechanisms, responsible for the ability of cells to adapt, performed bulk RNA sequencing with single-cell derived clones which showed different adaptability to TKIs and other stressors. In addition, we are utilizing agent-based mathematical modeling to further explore the dynamic changes of clonal heterogeneity in acquired resistance. We believe our ongoing experiments including the Clone-Tracer experiment, bulk RNA-seq data analysis and their validation, and mathematical modeling will provide a paradigm-wide shift in the understanding of acquired resistance from the current prevalent mutation-centric mechanism paradigm which may lead us to design evolutionary-informed therapy approaches to the resistance before it arises. Citation Format: Aobuli Xieraili, Matthew Froid, Andriy Marusyk. Epigenetic adaptability as a targetable trait underlying therapy resistance [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B029.

Update on Biology and Genomics of Adrenocortical Carcinomas: Rationale for Emerging Therapies.

The adrenal glands are paired endocrine organs that produce steroid hormones and catecholamines required for life. Adrenocortical carcinoma (ACC) is a rare and often fatal cancer of the peripheral domain of the gland, the adrenal cortex. Recent research in adrenal development, homeostasis, and disease have refined our understanding of the cellular and molecular programs controlling cortical growth and renewal, uncovering crucial clues into how physiologic programs are hijacked in early and late stages of malignant neoplasia. Alongside these studies, genome-wide approaches to examine adrenocortical tumors have transformed our understanding of ACC biology, and revealed that ACC is composed of distinct molecular subtypes associated with favorable, intermediate, and dismal clinical outcomes. The homogeneous transcriptional and epigenetic programs prevailing in each ACC subtype suggest likely susceptibility to any of a plethora of existing and novel targeted agents, with the caveat that therapeutic response may ultimately be limited by cancer cell plasticity. Despite enormous biomedical research advances in the last decade, the only potentially curative therapy for ACC to date is primary surgical resection, and up to 75% of patients will develop metastatic disease refractory to standard-of-care adjuvant mitotane and cytotoxic chemotherapy. A comprehensive, integrated, and current bench-to-bedside understanding of our field's investigations into adrenocortical physiology and neoplasia is crucial to developing novel clinical tools and approaches to equip the one-in-a-million patient fighting this devastating disease.

Epithelial-to-mesenchymal transition promotes immune escape by inducing CD70 in non-small cell lung cancer

IntroductionEpithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy. MethodsWe evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55). ResultsWe uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3+ and CD8+ T-cell infiltration and increased T-cell exhaustion markers. ConclusionOur results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.

OVOL1 inhibits breast cancer cell invasion by enhancing the degradation of TGF-\u03b2 type I receptor

Ovo-like transcriptional repressor 1 (OVOL1) is a key mediator of epithelial lineage determination and mesenchymal–epithelial transition (MET). The cytokines transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMP) control the epithelial–mesenchymal plasticity (EMP) of cancer cells, but whether this occurs through interplay with OVOL1 is not known. Here, we show that OVOL1 is inversely correlated with the epithelial–mesenchymal transition (EMT) signature, and is an indicator of a favorable prognosis for breast cancer patients. OVOL1 suppresses EMT, migration, extravasation, and early metastatic events of breast cancer cells. Importantly, BMP strongly promotes the expression of OVOL1, which enhances BMP signaling in turn. This positive feedback loop is established through the inhibition of TGF-β receptor signaling by OVOL1. Mechanistically, OVOL1 interacts with and prevents the ubiquitination and degradation of SMAD family member 7 (SMAD7), which is a negative regulator of TGF-β type I receptor stability. Moreover, a small-molecule compound 6-formylindolo(3,2-b)carbazole (FICZ) was identified to activate OVOL1 expression and thereby antagonizing (at least in part) TGF-β-mediated EMT and migration in breast cancer cells. Our results uncover a novel mechanism by which OVOL1 attenuates TGF-β/SMAD signaling and maintains the epithelial identity of breast cancer cells.

H3K4 demethylase KDM5B regulates cancer cell identity and epigenetic plasticity.

The H3K4 demethylase KDM5B is overexpressed in multiple cancer types, and elevated expression levels of KDM5B is associated with decreased survival. However, the underlying mechanistic contribution of dysregulated expression of KDM5B and H3K4 demethylation in cancer is poorly understood. Our results show that loss of KDM5B in multiple types of cancer cells leads to increased proliferation and elevated expression of cancer stem cell markers. In addition, we observed enhanced tumor formation following KDM5B depletion in a subset of representative cancer cell lines. Our findings also support a role for KDM5B in regulating epigenetic plasticity, where loss of KDM5B in cancer cells with elevated KDM5B expression leads to alterations in activity of chromatin states, which facilitate activation or repression of alternative transcriptional programs. In addition, we define KDM5B-centric epigenetic and transcriptional patterns that support cancer cell plasticity, where KDM5B depleted cancer cells exhibit altered epigenetic and transcriptional profiles resembling a more primitive cellular state. This study also provides a resource for evaluating associations between alterations in epigenetic patterning upon depletion of KDM5B and gene expression in a diverse set of cancer cells.

Cancer Cell Phenotype Plasticity as a Driver of Immune Escape in Melanoma.

Immunotherapies blocking negative immune checkpoints are now approved for the treatment of a growing number of cancers. However, even in metastatic melanoma, where sustained responses are observed, a significant number of patients still do not respond or display resistance. Increasing evidence indicates that non-genetic cancer cell-intrinsic alterations play a key role in resistance to therapies and immune evasion. Cancer cell plasticity, mainly associated with the epithelial-to-mesenchymal transition in carcinoma, relies on transcriptional, epigenetic or translational reprogramming. In melanoma, an EMT-like dedifferentiation process is characterized by the acquisition of invasive or neural crest stem cell-like features. Herein, we discuss recent findings on the specific roles of phenotypic reprogramming of melanoma cells in driving immune evasion and resistance to immunotherapies. The mechanisms by which dedifferentiated melanoma cells escape T cell lysis, mediate T cell exclusion or remodel the immune microenvironment will be detailed. The expanded knowledge on tumor cell plasticity in melanoma should contribute to the development of novel therapeutic combination strategies to further improve outcomes in this deadly metastatic cancer.

Activation of Drp1 promotes fatty acids-induced metabolic reprograming to potentiate Wnt signaling in colon cancer.

Cancer cells are known for their ability to adapt variable metabolic programs depending on the availability of specific nutrients. Our previous studies have shown that uptake of fatty acids alters cellular metabolic pathways in colon cancer cells to favor fatty acid oxidation. Here, we show that fatty acids activate Drp1 to promote metabolic plasticity in cancer cells. Uptake of fatty acids (FAs) induces mitochondrial fragmentation by promoting ERK-dependent phosphorylation of Drp1 at the S616 site. This increased phosphorylation of Drp1 enhances its dimerization and interaction with Mitochondrial Fission Factor (MFF) at the mitochondria. Consequently, knockdown of Drp1 or MFF attenuates fatty acid-induced mitochondrial fission. In addition, uptake of fatty acids triggers mitophagy via a Drp1- and p62-dependent mechanism to protect mitochondrial integrity. Moreover, results from metabolic profiling analysis reveal that silencing Drp1 disrupts cellular metabolism and blocks fatty acid-induced metabolic reprograming by inhibiting fatty acid utilization. Functionally, knockdown of Drp1 decreases Wnt/β-catenin signaling by preventing fatty acid oxidation-dependent acetylation of β-catenin. As a result, Drp1 depletion inhibits the formation of tumor organoids in vitro and xenograft tumor growth in vivo. Taken together, our study identifies Drp1 as a key mediator that connects mitochondrial dynamics with fatty acid metabolism and cancer cell signaling.

Redox-Based Strategy for Selectively Inducing Energy Crisis Inside Cancer Cells: An Example of Modifying Dietary Curcumin to Target Mitochondria.

Reprograming of energy metabolism is a major hallmark of cancer, but its effective intervention is still a challenging task due to metabolic heterogeneity and plasticity of cancer cells. Herein, we report a general redox-based strategy for meeting the challenge. The strategy was exemplified by a dietary curcumin analogue (MitoCur-1) that was designed to target mitochondria (MitoCur-1). By virtue of its electrophilic and mitochondrial-targeting properties, MitoCur-1 generated reactive oxygen species (ROS) more effectively and selectively in HepG2 cells than in L02 cells via the inhibition of mitochondrial antioxidative thioredoxin reductase 2 (TrxR2). The ROS generation preferentially mediated the energy crisis of HepG2 cells in a dual-inhibition fashion against both mitochondrial and glycolytic metabolisms, which could hit the metabolic plasticity of HepG2 cells. The ROS-dependent energy crisis also allowed its preferential killing of HepG2 cells (IC50 = 1.4 μM) over L02 cells (IC50 = 9.1 μM), via induction of cell-cycle arrest, apoptosis and autophagic death, and its high antitumor efficacy in vivo, in nude mice bearing HepG2 tumors (15 mg/kg). These results highlight that inhibiting mitochondrial TrxR2 to produce ROS by electrophiles is a promising redox-based strategy for the effective intervention of cancer cell energy metabolic reprograming.

Metabolic profiling of prostate cancer in skeletal microenvironments identifies G6PD as a key mediator of growth and survival.

The spread of cancer to bone is invariably fatal, with complex cross-talk between tumor cells and the bone microenvironment responsible for driving disease progression. By combining in silico analysis of patient datasets with metabolomic profiling of prostate cancer cells cultured with bone cells, we demonstrate the changing energy requirements of prostate cancer cells in the bone microenvironment, identifying the pentose phosphate pathway (PPP) as elevated in prostate cancer bone metastasis, with increased expression of the PPP rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD) associated with a reduction in progression-free survival. Genetic and pharmacologic manipulation demonstrates that G6PD inhibition reduces prostate cancer growth and migration, associated with changes in cellular redox state and increased chemosensitivity. Genetic blockade of G6PD in vivo results in reduction of tumor growth within bone. In summary, we demonstrate the metabolic plasticity of prostate cancer cells in the bone microenvironment, identifying the PPP and G6PD as metabolic targets for the treatment of prostate cancer bone metastasis.

Dissecting Tumor Growth: The Role of Cancer Stem Cells in Drug Resistance and Recurrence.

Simple SummaryCancer is one of the most debated problems all over the world. Cancer stem cells are considered responsible of tumor initiation, metastasis, drug resistance, and recurrence. This subpopulation of cells has been found into the tumor bulk and showed the capacity to self-renew, differentiate, up to generate a new tumor. In the last decades, several studies have been set on the molecular mechanisms behind their specific characteristics as the Wnt/β-catenin signaling, Notch signaling, Hedgehog signaling, transcription factors, etc. The most powerful part of CSCs is represented by the niches as “promoter” of their self-renewal and “protector” from the common oncological treatment as chemotherapy and radiotherapy. In our review article we highlighted the primary mechanisms involved in CSC tumorigenesis for the setting of further targets to control the metastatic process.Emerging evidence suggests that a small subpopulation of cancer stem cells (CSCs) is responsible for initiation, progression, and metastasis cascade in tumors. CSCs share characteristics with normal stem cells, i.e., self-renewal and differentiation potential, suggesting that they can drive cancer progression. Consequently, targeting CSCs to prevent tumor growth or regrowth might offer a chance to lead the fight against cancer. CSCs create their niche, a specific area within tissue with a unique microenvironment that sustains their vital functions. Interactions between CSCs and their niches play a critical role in regulating CSCs’ self-renewal and tumorigenesis. Differences observed in the frequency of CSCs, due to the phenotypic plasticity of many cancer cells, remain a challenge in cancer therapeutics, since CSCs can modulate their transcriptional activities into a more stem-like state to protect themselves from destruction. This plasticity represents an essential step for future therapeutic approaches. Regarding self-renewal, CSCs are modulated by the same molecular pathways found in normal stem cells, such as Wnt/β-catenin signaling, Notch signaling, and Hedgehog signaling. Another key characteristic of CSCs is their resistance to standard chemotherapy and radiotherapy treatments, due to their capacity to rest in a quiescent state. This review will analyze the primary mechanisms involved in CSC tumorigenesis, with particular attention to the roles of CSCs in tumor progression in benign and malignant diseases; and will examine future perspectives on the identification of new markers to better control tumorigenesis, as well as dissecting the metastasis process.

Quantifying the Patterns of Metabolic Plasticity and Heterogeneity along the Epithelial-Hybrid-Mesenchymal Spectrum in Cancer.

Cancer metastasis is the leading cause of cancer-related mortality and the process of the epithelial-to-mesenchymal transition (EMT) is crucial for cancer metastasis. Both partial and complete EMT have been reported to influence the metabolic plasticity of cancer cells in terms of switching among the oxidative phosphorylation, fatty acid oxidation and glycolysis pathways. However, a comprehensive analysis of these major metabolic pathways and their associations with EMT across different cancers is lacking. Here, we analyse more than 180 cancer cell datasets and show the diverse associations of these metabolic pathways with the EMT status of cancer cells. Our bulk data analysis shows that EMT generally positively correlates with glycolysis but negatively with oxidative phosphorylation and fatty acid metabolism. These correlations are also consistent at the level of their molecular master regulators, namely AMPK and HIF1α. Yet, these associations are shown to not be universal. The analysis of single-cell data for EMT induction shows dynamic changes along the different axes of metabolic pathways, consistent with general trends seen in bulk samples. Further, assessing the association of EMT and metabolic activity with patient survival shows that a higher extent of EMT and glycolysis predicts a worse prognosis in many cancers. Together, our results reveal the underlying patterns of metabolic plasticity and heterogeneity as cancer cells traverse through the epithelial–hybrid–mesenchymal spectrum of states.

Androgen receptor-mediated transcriptional repression targets cell plasticity in prostate cancer.

Androgen receptor (AR) signaling remains the key therapeutic target in the management of hormone‐naïve‐advanced prostate cancer (PCa) and castration‐resistant PCa (CRPC). Recently, landmark molecular features have been reported for CRPC, including the expression of constitutively active AR variants that lack the ligand‐binding domain. Besides their role in CRPC, AR variants lead to the expression of genes involved in tumor progression. However, little is known about the specificity of their mode of action compared with that of wild‐type AR (AR‐WT). We performed AR transcriptome analyses in an androgen‐dependent PCa cell line as well as cross‐analyses with publicly available RNA‐seq datasets and established that transcriptional repression capacity that was marked for AR‐WT was pathologically lost by AR variants. Functional enrichment analyses allowed us to associate AR‐WT repressive function to a panel of genes involved in cell adhesion and epithelial‐to‐mesenchymal transition. So, we postulate that a less documented AR‐WT normal function in prostate epithelial cells could be the repression of a panel of genes linked to cell plasticity and that this repressive function could be pathologically abrogated by AR variants in PCa.