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Colorectal cancer liver metastatic (CRCLM) tumours present as two main histopathological growth patterns (HGPs) including desmoplastic HGP (DHGP) and replacement HGP (RHGP). The DHGP tumours obtain their blood supply by sprouting angiogenesis, whereas the RHGP tumours utilize an alternative vascularisation known as vessel co-option. In vessel co-option, the cancer cells hijack the mature sinusoidal vessels to obtain blood supply. Vessel co-option has been reported as an acquired mechanism of resistance to anti-angiogenic treatment in CRCLM. Herein, we showed that inhibiting proprotein convertase subtilisin-like kexin type 9 (PCSK9) via clinically approved PCSK9-neutralizing antibody (Evolocumab) can boost the response of vessel co-opting tumours to anti-angiogenic therapy. Mechanistically, we found that PCSK9 inhibition downregulates runt related transcription factor-1 (RUNX1) expression levels in CRCLM cancer cells in vivo, which its expression positively correlates with the development of vessel co-option. Collectively, these results suggest that inhibiting PCSK9 is a promising way to improve the efficacy of anti-angiogenic therapy against vessel co-opting tumours in CRCLM.

Vessel co-option correlates with resistance against anti-angiogenic agents and chemotherapy in colorectal cancer liver metastasis (CRCLM). We previously identified higher intensity of neutrophils in the tumour microenvironment of vessel co-opting CRCLM lesions compared to their angiogenic counterparts. Herein, we demonstrated that over 50% of the neutrophils in vessel co-opting lesions are expressing pro-apoptotic markers including cleaved caspase-3 and poly (ADP-ribose) polymerase-1 (PARP-1). Our previous publications suggested upregulation of transforming growth factor-beta (TGFβ1) in the microenvironment of vessel co-option CRCLM. Therefore, we examined the effect of TGFβ1 on the expression of cleaved caspase-3 and PARP-1 in neutrophils in vitro. Significantly, we noticed the upregulation of pro-apoptotic markers upon exposure to TGFβ1. This finding might pave the way to determine the role of neutrophils in developing vessel co-option in CRCLM in the future.

Supplementary Figure 3. BTK and apoptosis.

p53 is a tumor suppressor that prevents the emergence of transformed cells by inducing apoptosis or senescence, among other responses. Its functions are regulated tightly by posttranslational modifications. Here we show that Bruton's tyrosine kinase (BTK) is a novel modulator of p53. We found that BTK is induced in response to DNA damage and p53 activation. BTK induction leads to p53 phosphorylation, which constitutes a positive feedback loop that increases p53 protein levels and enhances the transactivation of its target genes in response to stress. Inhibiting BTK reduced both p53-dependent senescence and apoptosis. Further, BTK expression also upregulated DNA damage signals and apoptosis. We conclude that despite being involved in oncogenic signals in blood malignancies, BTK has antineoplastic properties in other contexts, such as the enhancement of p53's tumor suppressor responses. Along with evidence that BTK expression correlates with good prognosis in some epithelial tumors, our findings may encourage a reevaluation of the clinical uses of BTK inhibitors in cancer therapy. Cancer Res; 76(18); 5405–14. ©2016 AACR.

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Colorectal cancer liver metastasis (CRCLM) is one of the deadliest cancers. CRCLM tumours have two distinct histopathological growth patterns (HGPs) including desmoplastic HGP (DHGP) and replacement HGP (RHGP). The DHGP tumours are angiogenic, while their RHGP counterparts are vessel co-opting. The patients with DHGP tumours have a better response to anti-angiogenic agents and chemotherapy, as well as the prognosis. To determine the influence of vitamin D supplementation in CRCLM, we analyzed the HGPs and the 5-year OS of CRCLM patients (n=106). Interestingly, we found an inverse correlation between vitamin D supplementation and the presence of RHGP tumours in CRCLM patients. Additionally, the 5-year OS of the patients that administered vitamin D was significantly higher. The cancer cells in RHGP lesions are characterized by direct contact with the hepatocytes, and this phenomenon enhances the motility of the cancer cells and facilitates their infiltration through liver parenchyma to co-opt the pre-existing vessels. Significantly, our in vitro data demonstrated the downregulation of motility markers in the co-cultured cancer cells with hepatocytes upon exposure to vitamin D. Altogether, this study highlights the role of vitamin D in CRCLM and provides a rationale to investigate the contribution of vitamin D supplementation to the prognosis of CRCLM patients.

Colorectal cancer liver metastatic (CRCLM) tumours present as two main histopathological growth patterns (HGPs) including desmoplastic HGP (DHGP) and replacement HGP (RHGP). The DHGP tumours obtain their blood supply by sprouting angiogenesis, whereas the RHGP tumours utilize an alternative vascularisation known as vessel co-option. In vessel co-option, the cancer cells hijack the mature sinusoidal vessels to obtain blood supply. Vessel co-option has been reported as an acquired mechanism of resistance to anti-angiogenic treatment in CRCLM. Here, we show the connection between the concentration of serum cholesterol and the development of vessel co-option in CRCLM. Our clinical data suggested that the elevation of serum cholesterol levels correlates with the risk of developing vessel co-opting tumours. Moreover, inhibition of the key modulators of cholesterol metabolism including HMGCR or PCSK9 attenuated the development of CRCLM tumours, as well as vessel co-option in vivo. Altogether, our data uncovered the importance of cholesterol in the development of vessel co-option tumours and demonstrated PCSK9 and HMGCR inhibitors as promising strategies to mitigate the development of vessel co-option tumours in CRCLM.

Vessel co-option is an alternative strategy by which tumour cells vascularize and gain access to nutrients to support tumour growth, survival and metastasis. In vessel co-option, the cancer cells move towards the pre-existing vasculature and hijack them. Vessel co-option is adopted by a wide range of human tumours including colorectal cancer liver metastases (CRCLM) and is responsible for the effectiveness of treatment in CRCLM. Furthermore, vessel co-option is an intrinsic feature and an acquired mechanism of resistance to anti-angiogenic treatment. In this review, we describe the microenvironment, the molecular players, discovered thus far of co-opting CRCLM lesions and propose a theoretical model. We also highlight key unanswered questions that are critical to improving our understanding of CRCLM vessel co-option and for the development of effective approaches for the treatment of co-opting tumours.

The outbreak of the novel coronavirus disease 2019 (COVID-19) has emerged as one of the biggest global health threats worldwide. As of October 2020, more than 44 million confirmed cases and more than 1,160,000 deaths have been reported globally, and the toll is likely to be much higher before the pandemic is over. There are currently little therapeutic options available and new potential targets are intensively investigated. Recently, Bruton tyrosine kinase (BTK) has emerged as an interesting candidate. Elevated levels of BTK activity have been reported in blood monocytes from patients with severe COVID-19, compared with those from healthy volunteers. Importantly, various studies confirmed empirically that administration of BTK inhibitors (acalabrutinib and ibrutinib) decreased the duration of mechanical ventilation and mortality rate for hospitalized patients with severe COVID-19. Herein, we review the current information regarding the role of BTK in severe acute respiratory syndrome coronavirus 2 infections and the suitability of its inhibitors as drugs to treat COVID-19. The use of BTK inhibitors in the management of COVID-19 shows promise in reducing the severity of the immune response to the infection and thus mortality. However, BTK inhibition may be contributing in other ways to inhibit the effects of the virus and this will need to be carefully studied.

Colorectal cancer liver metastases (CRCLM) that present with a replacement histopathological growth pattern (HGP) are resistant to neoadjuvant anti-angiogenic therapy. Surrogate biomarkers are not available to preoperatively identify patients with these tumors. Here we identify differentially expressed genes between CRCLM with a replacement HGP and those with a desmoplastic HGP using RNA sequencing. We demonstrate that LOXL4 is transcriptionally upregulated in replacement HGP CRCLM compared with desmoplastic HGP CRCLM and the adjacent normal liver. Interestingly, lysyl oxidase-like 4 (LOXL4) protein was expressed by neutrophils present in the tumor microenvironment in replacement HGP CRCLM. We further demonstrate that LOXL4 expression is higher in circulating neutrophils of cancer patients compared with healthy control patients and its expression can be induced by stimulation with lipopolysaccharide and TNF-α. Our study is the first to show the expression of LOXL4 in neutrophils and reveals the potential for LOXL4-expressing neutrophils to support the replacement HGP phenotype and to serve as a surrogate biomarker for this subtype of CRCLM. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Collagen type XI alpha 1 (COL11A1) is a novel biomarker associated with cisplatin resistance in ovarian cancer. However, the mechanisms underlying how COL11A1 confers cisplatin resistance in ovarian cancer are poorly understood. We identified that fatty acid β-oxidation (FAO) is upregulated by COL11A1 in ovarian cancer cells and that COL11A1-driven cisplatin resistance can be abrogated by inhibition of FAO. Furthermore, our results demonstrate that COL11A1 also enhances the expression of proteins involved in fatty acid synthesis. Interestingly, COL11A1-induced upregulation of fatty acid synthesis and FAO is modulated by the same signaling molecules. We identified that binding of COL11A1 to its receptors, α1β1 integrin and discoidin domain receptor 2 (DDR2), activates Src-Akt-AMPK signaling to increase the expression of both fatty acid synthesis and oxidation enzymes, although DDR2 seems to be the predominant receptor. Inhibition of fatty acid synthesis downregulates FAO despite the presence of COL11A1, suggesting that fatty acid synthesis might be a driver of FAO in ovarian cancer cells. Taken together, our results suggest that COL11A1 upregulates fatty acid metabolism in ovarian cancer cells in a DDR2-Src-Akt-AMPK dependent manner. Therefore, we propose that blocking FAO might serve as a promising therapeutic target to treat ovarian cancer, particularly cisplatin-resistant recurrent ovarian cancers which typically express high levels of COL11A1.

Colorectal cancer liver metastases (CRCLM) have two major histopathological growth patterns (HGPs) including angiogenic desmoplastic HGP (DHGP) and non-angiogenic replacement HGP (RHGP). The RHGP lesions obtain their blood supply through vessel co-option, where the cancer cells hijack the pre-existing blood vessels of the surrounding liver tissue. Consequently, anti-angiogenic therapies are less efficacious in CRCLM patients with RHGP lesions. Recently, we identified a positive correlation between the expression of Angiopoietin1 (Ang1) and the development of vessel co-opted CRCLM lesions in vivo. However, the mechanisms underlying Ang1 upregulation in vessel co-opting CRCLM lesions are unclear. Herein, we demonstrated that transforming growth factor β1 (TGFβ1) modulates the expression of Ang1 in hepatocytes in vitro. Significantly, pharmaceutical inhibition of integrin alpha-5/beta-1 (ITGα5β1) through ATN-161 impaired TGFβ1-dependent Ang1 expression in vitro and in vivo. Moreover, blocking ITGα5β1 attenuated the formation of vessel co-opting lesions. Furthermore, treatment with ATN-161 significantly improved survival in tumour-bearing mice. Taken together, our results suggest the molecular mechanism of Ang1 upregulation in vessel co-opting CRCLM and targeting this pathway may serve as promising therapeutic strategy to overcome the development of vessel co-option in CRCLM.

Colorectal cancer liver metastasis (CRCLM) is one of the deadliest cancers. CRCLM tumours have two distinct histopathological growth patterns (HGPs) including desmoplastic HGP (DHGP) and replacement HGP (RHGP). The DHGP tumours are angiogenic, while their RHGP counterparts are vessel co-opting. The patients with RHGP tumours showed poor response to anti-angiogenic agents, as well as a worse prognosis. Herein, we conducted a retrospective cohort study that comprised 106 CRCLM patients to examine the effect of vitamin D supplementation on the HGPs of the tumours and the 5-year overall survival (OS). Interestingly, we found an inverse correlation between vitamin D supplementation and the presence of RHGP tumours in CRCLM patients. Additionally, the population who used vitamin D supplementation had significantly better 5-year OS. Moreover, our in vivo results suggested that vitamin D supplementation significantly improves the response of CRCLM tumours to anti-angiogenic therapy. Mechanistically, we found that vitamin D can suppress cancer cell motility, which is essential for the development of vessel co-option tumours and resistance to anti-angiogenic therapy. Collectively, this study suggests vitamin D supplementation is a promising way to attenuate resistance to anti-angiogenic therapy and improve the prognosis of CRCLM patients. Citation Format: Miran Rada, Lucyna Krzywon, Audrey Kapelanski-Lamoureux, Stephanie Petrillo, Anthoula Lazaris, Peter Metrakos. Vitamin D supplementation attenuates resistance to anti-angiogenic therapy in colorectal cancer liver metastases. [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 4601.

Colorectal cancer liver metastatic (CRCLM) tumours present as two main histopathological growth patterns (HGPs) including desmoplastic HGP (DHGP) and replacement HGP (RHGP). The DHGP tumours obtain their blood supply by sprouting angiogenesis, whereas the RHGP tumours utilize an alternative vascularisation known as vessel co-option. In vessel co-option, the cancer cells hijack the mature sinusoidal vessels to obtain blood supply. Vessel co-option has been reported as an acquired mechanism of resistance to anti-angiogenic treatment in CRCLM. Herein, we show the connection between serum cholesterol concentration and vessel co-option development in CRCLM. Our clinical data suggested that the elevation of serum cholesterol levels correlates with the risk of developing vessel co-opting tumours. Moreover, inhibition of PCSK9, the key modulator of cholesterol metabolism, significantly attenuated the development of vessel co-opting CRCLM tumours and sensitized the tumours to anti-angiogenic therapy in vivo. Altogether, these data suggest the importance of cholesterol in the development of vessel co-option tumours and propose that inhibiting PCSK9 is a promising strategy to overcome resistance to anti-angiogenic therapy in CRCLM. Citation Format: Miran Rada, Lucyna Krzywon, Audrey Kapelanski-Lamoureux, Stephanie Petrillo, Andrew Reynolds, Anthoula Lazaris, Nabil Seidah, Peter Metrakos. Inhibition of pcsk9 impairs the development of vessel co-option and potentiates anti-angiogenic therapy in colorectal cancer liver metastases [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 1741.

Vessel co-option is associated with resistance against anti-angiogenic therapy in colorectal cancer liver metastases (CRCLM). Vessel co-opted lesions are characterized by highly motile cancer cells that move toward and along the pre-existing vessels in the surrounding non-malignant tissue and hijack them to gain access to nutrient. In order to access the sinusoidal vessels, the cancer cells must displace the hepatocytes and occupy their space. However, the mechanisms underlying this displacement are unknown. Herein, we examined the involvement of apoptosis, motility, epithelial-mesenchymal transition (EMT) and autophagy pathways in hepatocyte’s displacement by cancer cells. We demonstrated that cancer cells induce the expression of the proteins that associated with upregulation of apoptosis, motility and EMT in adjacent hepatocytes in vitro and in vivo. Accordingly, we observed upregulation of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and Actin Related Protein 2/3 (ARP2/3) in adjacent hepatocytes to cancer cells, while we noticed lower expression levels of E-cadherin. Importantly, Runt-related transcription factor 1 (RUNX1) knockdown in the cancer cells has impaired their function towards adjacent hepatocytes in vitro and in vivo. Altogether, our data indicating that cancer cells may exploit various mechanisms to displace the hepatocytes and access to the sinusoidal vessels to establish vessel co-option. Citation Format: Miran Rada, Anthoula Lazaris, Peter Metrakos. Cancer cells promote phenotypic alterations in adjacent hepatocytes to facilitate vessel co-option in colorectal cancer liver metastasis [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 1580.

Vessel co-option in colorectal cancer liver metastases (CRCLM) has been recognized as one of the mechanistic pathways that contribute to resistance against anti-angiogenic therapy. In vessel co-opted CRCLM lesions, the cancer cells are highly motile that move toward and along the pre-existing sinusoidal vessels and hijack them to gain access to nutrient. The movement of cancer cells is accompanied by replacement of the hepatocytes. However, the molecular mechanisms by which this replacement occurs are unclear yet. To examine the involvement of apoptosis in hepatocytes replacement by cancer cells in co-opted lesions, we conducted immunohistochemical staining for chemonaïve CRCLM specimens using pro-apoptotic markers antibody, such as cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase-1 (PARP-1). The results suggested overexpression of pro-apoptotic markers in liver parenchyma of co-opted lesions compared to angiogenic lesions, specifically the hepatocytes that are in close proximity to the cancer cells. Importantly, co-culturing hepatocytes with colorectal cancer cells induced overexpression of pro-apoptotic markers in the hepatocytes. Altogether, these results propose that cancer cells could exploit apoptosis to replace the hepatocytes and establish vessel co-option in CRCLM.

Resistance to anti-angiogenic therapy is a major challenge in the treatment of colorectal cancer liver metastases (CRCLMs). Vessel co-option has been identified as a key contributor to anti-angiogenic therapy resistance in CRCLMs. Recently, we identified a positive correlation between the expression of Angiopoietin1 (Ang1) in the liver and the development of vessel co-opting CRCLM lesions in vivo. However, the mechanisms underlying its stimulation of vessel co-option are unclear. Herein, we demonstrated Ang1 as a positive regulator of actin-related protein 2/3 (ARP2/3) expression in cancer cells, in vitro and in vivo, which is known to be essential for the formation of vessel co-option in CRCLM. Significantly, Ang1-dependent ARP2/3 expression was impaired in the cancer cells upon Tie2 or PI3K/AKT inhibition in vitro. Taken together, our results suggest novel mechanisms by which Ang1 confers the development of vessel co-option in CRCLM, which, targeting this pathway, may serve as promising therapeutic targets to overcome the development of vessel co-option in CRCLM.

Vessel co-option is correlated with resistance against anti-angiogenic therapy in colorectal cancer liver metastases (CRCLM). Vessel co-opting lesions are characterized by highly motile cancer cells that move toward and along the pre-existing vessels in the surrounding nonmalignant tissue and co-opt them to gain access to nutrients. To access the sinusoidal vessels, the cancer cells in vessel co-opting lesions must displace the hepatocytes and occupy their space. However, the mechanisms underlying this displacement are unknown. Herein, we examined the involvement of apoptosis, autophagy, motility, and epithelial–mesenchymal transition (EMT) pathways in hepatocyte displacement by cancer cells. We demonstrate that cancer cells induce the expression of the proteins that are associated with the upregulation of apoptosis, motility, and EMT in adjacent hepatocytes in vitro and in vivo. Accordingly, we observe the upregulation of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and actin-related protein 2/3 (ARP2/3) in adjacent hepatocytes to cancer cell nests, while we notice a downregulation of E-cadherin. Importantly, the knockdown of runt-related transcription factor 1 (RUNX1) in cancer cells attenuates the function of cancer cells in hepatocytes alterations in vitro and in vivo. Altogether, our data suggest that cancer cells exploit various mechanisms to displace hepatocytes and access the sinusoidal vessels to establish vessel co-option.

Although, cisplatin resistance is a major challenge in the treatment of ovarian cancer, the precise mechanisms underlying cisplatin resistance are not fully understood. Collagen type XI alpha 1 (COL11A1), a gene encoding a minor fibrillar collagen of the extracellular matrix, is identified as one of the most upregulated genes in cisplatin-resistant ovarian cancer and recurrent ovarian cancer. However, the exact functions of COL11A1 in cisplatin resistance are unknown. Here we demonstrate that COL11A1 binds to integrin α1β1 and discoidin domain receptor 2 (DDR2) and activates downstream signaling pathways to inhibit cisplatin-induced apoptosis in ovarian cancer cells. Mechanistically, we show that COL11A1 activates Src-PI3K/Akt-NF-kB signaling to induce the expression of three inhibitor apoptosis proteins (IAPs), including XIAP, BIRC2, and BIRC3. Genetic and pharmacological inhibition of XIAP, BIRC2, and BIRC3 is sufficient to restore cisplatin-induced apoptosis in ovarian cancer cells in the presence of COL11A1 in ovarian cancer cells and xenograft mouse models, respectively. We also show that the components of COL11A1- integrin α1β1/DDR2- Src-PI3K/Akt-NF-kB-IAP signaling pathway serve as poor prognosis markers in ovarian cancer patients. Taken together, our results suggest novel mechanisms by which COL11A1 confers cisplatin resistance in ovarian cancer. Our study also uncovers IAPs as promising therapeutic targets to reduce cisplatin resistance in ovarian cancer, particularly in recurrent ovarian cancer expressing high levels of COL11A1.

One of the hallmarks of aging is the progressive accumulation of senescent cells in organisms, which has been proposed to be a contributing factor to age‐dependent organ dysfunction. We recently reported that Bruton's tyrosine kinase (BTK) is an upstream component of the p53 responses to DNA damage. BTK binds to and phosphorylates p53 and MDM2, which results in increased p53 activity. Consistent with this, blocking BTK impairs p53‐induced senescence. This suggests that sustained BTK inhibition could have an effect on organismal aging by reducing the presence of senescent cells in tissues. Here, we show that ibrutinib, a clinically approved covalent inhibitor of BTK, prolonged the maximum lifespan of a Zmpste24−/− progeroid mice, which also showed a reduction in general age‐related fitness loss. Importantly, we found that certain brain functions were preserved, as seen by reduced anxiety‐like behaviour and better long‐term spatial memory. This was concomitant to a decrease in the expression of specific markers of senescence in the brain, which confirms a lower accumulation of senescent cells after BTK inhibition. Our data show that blocking BTK has a modest increase in lifespan in Zmpste24−/− mice and protects them from a decline in brain performance. This suggests that specific inhibitors could be used in humans to treat progeroid syndromes and prevent the age‐related degeneration of organs such as the brain., We showed that blocking Bruton's tyrosine kinase (BTK) dampens p53 activity and the induction of senescence in vitro. Here, we show that BTK inhibitors increase maximum lifespan and healthspan of progeroid mice. Moreover, they prevent the deterioration of certain cognitive functions and reduce the accumulation of senescent cells in the brain. This suggests that clinically available BTK inhibitors could ameliorate some features of ageing in susceptible patients.

Vessel co-option in colorectal cancer liver metastases (CRCLM) has been recognized as one of the mechanistic pathways of resistance against anti-angiogenic therapy. The cancer cells are highly motile in co-opted lesions, which move toward and along the pre-existing sinusoidal vessels and hijack them to gain access to nutrient. The movement of cancer cells is accompanied by displacement of the hepatocytes. However, the molecular mechanisms underlying this displacement are unclear yet. To examine whether apoptosis involved in hepatocytes displacement by cancer cells in co-opted lesions, we performed immunohistochemical staining for pro-apoptotic markers, such as cleaved caspase-3 and cleaved PARP-1. We observed overexpression of pro-apoptotic markers in liver parenchyma of co-opted lesions compared to angiogenic lesions, specifically the hepatocytes that are in close proximity to the cancer cells. In vitro, we found that culturing hepatocytes with either colorectal cancer cells or conditioned media of co-opted CRCLM organoids induces apoptosis. Importantly, our results also suggested proprotein convertase subtilisin/kexin type 9 (PCSK-9 or PC-9) as a potential mediator of cancer cells-driven hepatocytes apoptosis. Altogether, these results confirm that cancer cells exploit apoptosis to establish vessel co-option in CRCLM. Citation Format: Miran Rada, Migmar Tsamchoe, Audrey Kapelanski-Lamoureux, Jessica Bloom, Stephanie Petrillo, Sébastien Tabariès, Diane H. Kim, Peter Younan, Alex Gregorieff, Peter Siegel, Anthoula Lazaris, Peter Metrakos. Cancer cells induce apoptosis in hepatocytes as one of the mechanisms to displace hepatocytes in vessel co-opted colorectal cancer liver metastases [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 1927.

Colorectal cancer liver metastasis (CRCLM) has two major morphological growth pattern subtypes including desmoplastic (DHGP) and replacement (RHGP). The DHGP tumors depend on angiogenesis for their growth. Conversely, RHGP tumors are non-angiogenic and obtain their blood supply through vessel co-option, which the cancer cells hijacking pre-existing blood vessels of the surrounding liver tissue. Therefore, anti-angiogenic therapies have shown modest efficacy in CRCLM patients with RHGP lesions. In this study, we found overexpression of Runt Related Transcription Factor-1 (RUNX1) in the cancer cells of RHGP lesions. Upregulation of RUNX1 is positively correlated with cancer cell motility and epithelial mesenchymal transition (EMT) in vitro. Our results also showed that RUNX1 regulated by Transforming Growth Factor Beta-1 (TGFβ1) through TGFβ Receptor II (TGFβRII). Significantly, RUNX1 knockdown induced conversion RHGP lesions to DHGP in vivo. Collectively, this study suggests RUNX1 as a potential target therapy to overcome resistance to anti-angiogenic therapy in CRCLM. Citation Format: Miran Rada, Audrey Kapelanski-Lamoureux, Stephanie Petrillo, Sébastien Tabariès, Peter Siegel, Anthoula Lazaris, Peter Metrakos. Runt related transcription factor-1 (runx1) plays a central role in vessel co-option of colorectal cancer liver metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3935.

Many genes of the human genome display pleiotropic activity, playing an important role in two or more unrelated pathways. Surprisingly, some of these functions can even be antagonistic, often letting to divergent functional outcomes depending on microenviromental cues and tissue/cell type-dependent parameters. Lately, the Bruton’s tyrosine kinase (BTK) has emerged as one of such pleiotropic genes, with opposing effects in cancer pathways. While it has long been considered oncogenic in the context of B cell malignancies, recent data shows that BTK can also act as a tumour suppressor in other cells, as an essential member of the p53 and p73 responses to damage. Since BTK inhibitors are already being used clinically, it is important to carefully review these new findings in order to fully understand the consequences of blocking BTK activity in all the cells of the organism.

Bruton’s tyrosine kinase (BTK) is a key component of B cell receptor signalling. Because of this, BTK plays an important role in cell proliferation and survival in various B cell malignancies. However, in certain contexts, BTK can also have tumour suppressor functions. We have previously shown that BTK activates the p53 transcriptional activity by binding to and phosphorylating p53, as well as acting on MDM2 to reduce its inhibitory effects. This results in increased p53 functions, including enhanced cell death. Here, we report that BTK can also induce cell death and increase responses to DNA damage independently of p53. This is concomitant to the induction of p21, PUMA and MDM2, which are classic target genes of the p53 family of proteins. Our results show that these p53-independent effects of BTK are mediated through p73. Similar to what we observed in the p53 pathway, BTK can upregulate p73 after DNA damage and induce expression of its target genes, suggesting that BTK is a modulator of p73 functions and in the absence of p53. This effect allows BTK to have pro-apoptotic functions independently of its effects on the p53 pathway and thus play an important role in the DNA damage-related induction of apoptosis in the absence of p53. This provides a novel role of BTK in tumour suppression and contributes to the understanding of its complex pleiotropic functions

The introduction of nanomaterials into cells is an indispensable process for studies ranging from basic biology to clinical applications. To deliver foreign nanomaterials into living cells, traditionally endocytosis, viral and lipid nanocarriers or electroporation are mainly employed; however, they critically suffer from toxicity, inconsistent delivery, and low throughput and are time-consuming and labor-intensive processes. Here, we present a novel inertial microfluidic cell hydroporator capable of delivering a wide range of nanomaterials to various cell types in a single-step without the aid of carriers or external apparatus. The platform inertially focuses cells into the channel center and guides cells to collide at a T-junction. Controlled compression and shear forces generate transient membrane discontinuities that facilitate passive diffusion of external nanomaterials into the cell cytoplasm while maintaining high cell viability. This hydroporation method shows superior delivery efficiency, is high-throughput, and has high controllability; moreover, its extremely simple and low-cost operation provides a powerful and practical strategy in the applications of cellular imaging, biomanufacturing, cell-based therapies, regenerative medicine, and disease diagnosis.

p53 is a tumour suppressor that is activated in response to various types of stress. It is regulated by a complex pattern of over 50 different post-translational modifications, including ubiquitination by the E3 ligase MDM2, which leads to its proteasomal degradation. We have previously reported that expression of Bruton's Tyrosine Kinase (BTK) induces phosphorylation of p53 at the N-terminus, including Serine 15, and increases its protein levels and activity. The mechanisms involved in this process are not completely understood. Here, we show that BTK also increases MDM2 and is necessary for MDM2 upregulation after DNA damage, consistent with what we have shown for other p53 target genes. Moreover, we found that BTK binds to MDM2 on its PH domain and induces its phosphorylation. This suggested a negative regulation of MDM2 functions by BTK, supported by the fact BTK expression rescued the inhibitory effects of MDM2 on p53 transcriptional activity. Indeed, we observed that BTK mediated the loss of the ubiquitination activity of MDM2, a process that was dependent on the phosphorylation functions of BTK. Our data together shows that the kinase activity of BTK plays an important role in disrupting the MDM2-p53 negative feedback loop by acting at different levels, including binding to and inactivation of MDM2. This study provides a potential mechanism to explain how BTK modulates p53 functions.

Colorectal cancer liver metastases (CRCLM) that receive their blood supply via vessel co-option are associated with a poor response to anti-angiogenic therapy. Angiopoietins (Ang1 and Ang2) with their Tyrosine-protein kinase receptor (Tie2) have been shown to support vessel co-option. We demonstrate significantly higher expression of Ang1 in hepatocytes adjacent to the tumor region of human chemona&iuml, ve and treated co-opting (replacement histopathological growth patterns: RHGP) tumors. To investigate the role of the host Ang1 expression, Ang1 knockout (KO) mice were injected intra-splenically with metastatic MC-38 colon cancer cells that develop co-opting liver metastases. We observed a reduction in the number of liver metastases and interestingly, for the first time, the development of angiogenic driven desmoplastic (DHGP) liver metastases. In addition, in-vitro, knockout of Ang1 in primary hepatocytes inhibited viability, migration and invasion ability of MC-38 cells. We also demonstrate that Ang 1 alone promotes the migration and growth of both human and mouse colon cancer cell lines These results provide evidence that high expression of Ang1 in the host liver is important to support vessel co-option (RHGP lesions) and when inhibited, favours the formation of angiogenic driven liver metastases (DHGP lesions).

Colorectal carcinoma (CRC) remains the second leading cause of cancer death in the western world. Over 50% of CRC patients develop liver metastases (LM) and 90% will succumb to their disease. Liver resection of the LMs provides the only possibility of cure, but only 20% of colorectal cancer liver metastases (CRCLM) patients are resectable. The combination of angiogenic inhibitors (AI: anti-VEGF) with chemotherapy is the current form of treatment. Unfortunately, 65-70% of the patients continue on chemotherapy until resistance develops and then are treated with second, third and some times a fourth line of treatment, with an expected median overall survival of 24-28 months. We have no way of identifying those CRCLM patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. Bevacizumab). Recently we have identified two CRCLM histologic growth patterns (HGP) that predict treatment response and survival: 1) Desmoplastic (DHGP), a desmoplastic ring separating cancer cells from the liver parenchyma and lesions grow by angiogenesis; 2) Replacement or infiltrative (RHGP), tumor cells infiltrate the parenchymal cells in the liver as the lesions grow by co-opting the sinusoidal blood vessels between the liver cell plates. We showed that CRCLM patients with predominantly desmoplastic HGP metastasis receiving AIs plus chemotherapy have more than double the 5-year overall survival compared to patients with replacement HGP who have received the same treatment. In addition, our clinical data revealed that Angiogenic Inhibitors could negatively affect outcomes in patients with replacement HGPs. These non-angiogenic lesions do not respond to angiogenic inhibitors. To further our understanding of the molecular differences between the two HGPs we demonstrated by knocking out ARPC3 (Actin-related protein 2/3 complex subunit 3, involved in actin polymerization) in the human colon cancer cell line, HT-29, that cancer cell motility is a crucial process that regulates histological growth pattern in CRCLM. HT29 CRC cells injected directly into the mouse liver grow into replacement HGPs, while HT29s silenced for ARPC3 grow into desmoplastic HGPs lesions. However, the molecular mechanisms that regulate ARPC3 in CRCLM remain unknown. To further dissect the molecular mechanisms differentiating desmoplastic from replacement HGPs, we performed RNA-seq analysis of CRCLM lesions from chemonaïve patients. Our data revealed that both TGFβ1 and RUNX1 were upregulated in RHGP comparing to DHGP lesions. This has been further validated by immunoblotting and immunohistochemistry. Consistently, RUNX1 has been reported as a downstream of TGFβ1 and transcriptional factor for ARPC3. Collectively, our data suggests that TGFβ1 and RUNX1 contribute to the formation of infiltrative type of colorectal cancer liver metastases possibly through upregulation of ARPC3. Note: This abstract was not presented at the meeting. Citation Format: Miran Rada, Anthoula Lazaris, Stephanie Petrillo, Abdellatif Amri, Peter Metrakos. Deciphering the molecular mechanisms driving infiltrative histopathological type of colorectal cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5151.

p53 is a tumor suppressor that prevents the emergence of transformed cells by inducing apoptosis or senescence, among other responses. Its functions are regulated tightly by posttranslational modifications. Here we show that Bruton's tyrosine kinase (BTK) is a novel modulator of p53. We found that BTK is induced in response to DNA damage and p53 activation. BTK induction leads to p53 phosphorylation, which constitutes a positive feedback loop that increases p53 protein levels and enhances the transactivation of its target genes in response to stress. Inhibiting BTK reduced both p53-dependent senescence and apoptosis. Further, BTK expression also upregulated DNA damage signals and apoptosis. We conclude that despite being involved in oncogenic signals in blood malignancies, BTK has antineoplastic properties in other contexts, such as the enhancement of p53's tumor suppressor responses. Along with evidence that BTK expression correlates with good prognosis in some epithelial tumors, our findings may encourage a reevaluation of the clinical uses of BTK inhibitors in cancer therapy. Cancer Res; 76(18); 5405–14. ©2016 AACR.

Cisplatin resistance is the major challenge in the treatment of ovarian cancer, and it is crucial to develop novel therapeutic strategies to block cisplatin resistance. The purpose of this study was to identify new therapeutic targets for the treatment of cisplatin-resistant ovarian cancer. We previously identified collagen type XI alpha 1 (COL11A1) as a novel biomarker associated with cisplatin resistance in ovarian cancer. COL11A1 is expressed by cancer-associated fibroblasts (CAFs) adjacent to cancer cells as well as cisplatin-resistant ovarian cancer cell lines. Our data indicate that COL11A1 inhibits cisplatin-induced apoptosis in ovarian cancer cells, but not through altered gene expression of many of the well-established mechanisms of cisplatin resistance (i.e., transport, inactivation, DNA repair pathways). Rather, our proteomics data revealed that mitochondrial fatty acid beta-oxidation (FAO) was the most upregulated pathway by COL11A1 in ovarian cancer cells. To determine a link between COL11A1-driven cisplatin resistance and FAO, we knocked down COL11A1 in A2780CIS cisplatin-resistant ovarian cancer cells, a cell line expressing high endogenous levels of COL11A1. COL11A1 knockdown not only decreased the expression of genes involved in fatty acid beta-oxidation, such as CD36, CPT1A, ACAA2, HADHA, but also decreased fatty acid oxidation rate and increased cisplatin-induced apoptosis in A2780CIS ovarian cancer cells. In contrast, ovarian cancer cells became resistant to cisplatin-induced apoptosis and increased fatty acid beta-oxidation when exposed to COL11A1 protein or cocultured with COL11A1-overexpressing CAFs. Importantly, these cancer cells became more sensitive to the FAO inhibitor etomoxir. Collectively, our results suggest that COL11A1 confers cisplatin resistance by increasing FAO in ovarian cancer cells. Our study uncovers FAO as a promising therapeutic target to reduce cisplatin resistance in ovarian cancer, particularly in recurrent ovarian cancers that typically express high levels of COL11A1. Citation Format: Miran Rada, Jennifer Cha, Jessica Sage, Bo Zhou, Wei Yang, Sandra Orsulic, Dong-Joo Cheon. COL11A1 confers cisplatin resistance through fatty acid oxidation in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A16.

Ovarian cancer is the most lethal gynecological cancer and affects one in 70 females in the United States during their lifetime. Cisplatin resistance is the major challenge in the treatment of ovarian cancer but the underlying mechanisms are not fully understood. The purpose of this study is to develop novel therapeutic strategies to treat cisplatin-resistant ovarian cancer. We identified collagen type XI alpha 1 (COL11A1) as a novel biomarker associated with cisplatin resistance in ovarian cancer. COL11A1 is highly expressed by cancer-associated fibroblasts adjacent to cancer cells as well as A2780CIS cisplatin-resistant ovarian cancer cell line. Our data show that COL11A1 binds to DDR2 in ovarian cancer cells and inhibits cisplatin-induced apoptosis in ovarian cancer cells. To obtain mechanistic insights underlying COL11A1-driven cisplatin resistance, we performed Tandem Mass Tag proteomic analysis of A2780CIS ovarian cancer cells before and after COL11A1 knockdown. Our proteomics data revealed that mitochondrial fatty acid β-oxidation was the most upregulated pathway by COL11A1 in ovarian cancer cells. Mitochondrial fatty acid β-oxidation is the major pathway that breaks down fatty acids to produce excess ATP and NADPH to support cell survival. Several studies suggested the association of β-oxidation with tumor cell proliferation in various cancers. However, the role of β-oxidation in ovarian cancer chemoresistance is largely unknown. Our results show that COL11A1 enhances the expression of not only fatty acid receptor CD36, but also key enzymes of fatty acid β-oxidation, such as CPT1A, HADHB, ACSL1 and ACAA2. Significantly, COL11A1-mediated overexpression of these proteins was diminished in the presence of shRNA against DDR2.We also confirmed that ovarian cancer cells increase both fatty acid uptake and oxygen consumption rate in response to palmitate in the presence of COL11A1. Importantly, inhibition of fatty acid β-oxidation using shRNA against CPT1A attenuated the function of COL11A1 in cisplatin resistance. Taken together, our results suggest that COL11A1 confers cisplatin resistance by increasing fatty acid β-oxidation in ovarian cancer cells. Our study uncovers fatty acid β-oxidation as a promising therapeutic target to treat cisplatin-resistant ovarian cancer, particularly cisplatin-resistant recurrent ovarian cancers which typically express high levels of COL11A1. Citation Format: Miran Rada, Jennifer Cha, Jessica Sage, Bo Zhou, Wei Yang, Zahra Ashkavand, Kenneth Norman, Sandra Orsulic, Dong-Joo Cheon. Beta-oxidation inhibition as a novel therapy for cisplatin-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3526.

p53 is a critical tumor suppressor in humans. It functions mostly as a transcriptional factor and its activity is regulated by numerous post-translational modifications. Among different covalent modifications found on p53 the most controversial one is lysine methylation. We found that human G9a (hG9a) unlike its mouse orthologue (mG9a) potently stimulated p53 transcriptional activity. Both ectopic and endogenous hG9a augmented p53-dependent transcription of pro-apoptotic genes, including Bax and Puma, resulting in enhanced apoptosis and reduced colony formation. Significantly, shRNA-mediated knockdown of hG9a attenuated p53-dependent activation of Puma. On the molecular level, hG9a interacted with histone acetyltransferase, p300/CBP, resulting in increased histone acetylation at the promoter of Puma. The bioinformatics data substantiated our findings showing that positive correlation between G9a and p53 expression is associated with better survival of lung cancer patients. Collectively, this study demonstrates that depending on the cellular and organismal context, orthologous proteins may exert both overlapping and opposing functions. Furthermore, this finding has important ramifications on the use of G9a inhibitors in combination with genotoxic drugs to treat p53-positive tumors.

Ovarian cancer is the most lethal gynecological cancer, which affects one in 70 females in the United States during their lifetime. While cisplatin is the frontline treatment for ovarian cancer, chemoresistance severely limits the treatment success. The goal of this study is to identify novel therapeutic targets to overcome cisplatin resistance. We recently identified Collagen type XI alpha 1 (COL11A1) as a novel biomarker associated with cisplatin resistance in ovarian cancer. COL11A1 is a component of the tumor stroma and its expression is restricted to cancer-associated fibroblasts (CAFs) adjacent to tumor cells. We further showed that COL11A1 protects ovarian cancer cells from cisplatin-induced apoptosis, yet the underlying mechanism is largely unknown. To understand the molecular mechanism by which COL11A1 protects cancer cells from cisplatin-induced apoptosis, we overexpressed COL11A1 in ovarian cancer cells and performed RNA-Seq. Our RNA-Seq data revealed that COL11A1 did not change the levels of genes involved in cisplatin transport, inactivation, and DNA repair pathways in cancer cells, which are well-established mechanisms to confer cisplatin resistance. Rather, COL11A1 enhances the expression of the Inhibitors of Apoptosis Proteins (IAPs), including XIAP, BIRC2 (c-IAP1) and BIRC3 (c-IAP2). Using a full-length COL11A1 protein, genetic manipulation, and co-culture method, we confirmed that COL11A1 significantly increases the expression of IAPs upon cisplatin treatment. The inhibition of XIAP, BIRC2 and BIRC3 with an IAP antagonist BV6 attenuated the ability of COL11A1 to inhibit cisplatin-mediated apoptosis. Our data indicates that COL11A1 activates Akt, and Akt is known to phosphorylate XIAP at serine-87, which protects it from ubiquitination and degradation in response to cisplatin. Collectively, our data identify IAP as a mediator of COL11A1’s action in blocking cisplatin-induced apoptosis. Note: This abstract was not presented at the meeting. Citation Format: Miran Rada, Jennifer Cha, Jessica Sage, Sandra Orsulic, Dong-Joo Cheon. Collagen type XI alpha 1 confers cisplatin chemoresistance in ovarian cancer through inhibitor of apoptosis proteins (IAPs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2975. doi:10.1158/1538-7445.AM2017-2975

Despite an initial satisfactory response to platinum and taxane-based regimens, approximately 80% of ovarian cancer patients will succumb to chemoresistant recurrent disease. Thus, there is a dire need to understand the mechanism by which tumor cells develop resistance to subsequent therapies after first-line cisplatin treatment. We previously identified that collagen type XI, alpha 1 (COL11A1) is a novel biomarker associated with poor survival, metastasis, and chemoresistance in ovarian cancer. COL11A1 is expressed in a subset of α-SMA-positive cancer-associated fibroblasts (CAFs) adjacent to tumor cells. COL11A1 expression in peritumoral CAFs increases during ovarian cancer progression, with the highest expression in recurrent tumors. However, the functional significance of stromal COL11A1 in ovarian cancer progression and chemoresistance is largely unknown. To determine the role of stromal COL11A1 in chemoresistance, we generated COL11A1-overexpressing TRS3 ovarian fibroblasts, co-cultured them with A2780 and OVCAR3 ovarian cancer cells, and measured cancer cell sensitivity to cisplatin. Our data showed that ovarian cancer cells become resistant to cisplatin when cultured in direct contact with COL11A1-overexpressing fibroblasts as well as in fibroblast conditioned media. Furthermore, full-length COL11A1 protein, extracted from A204 cell line, increased cisplatin resistance in ovarian cancer cells, suggesting that COL11A1 is sufficient to induce chemoresistance. Interestingly, culture of cancer cells on full-length COL11A1 protein resulted in increased number of ALDHhigh cancer stem-like cells and activation of Akt, the principal mediator of pro-survival signaling. To further dissect the mechanism by which COL11A1 leads to cisplatin resistance in tumor cells, we performed RNA-seq analysis of tumor cells after COL11A1 overexpression. Our data showed that COL11A1 did not change the levels of genes involved in cisplatin transport and DNA repair pathway in cancer cells, which are well-established mechanisms to confer cisplatin resistance. Rather, COL11A1 increased the expression of pro-inflammatory genes, some of which are known to confer chemoresistance in ovarian cancer. Consistently, our Western data showed that COL11A1 activates NF-kB signaling in cancer cells, a central pathway regulating pro-inflammatory genes. Collectively, our data suggest that COL11A1 contributes to chemoresistance possibly due to an increased survival of ALDHhigh cancer stem-like cells through activation of pro-inflammatory signaling. Citation Format: Miran Rada, Catherine Eldred, Jessica Sage, Sandra Orsulic and Dong-Joo Cheon. COLLAGEN TYPE XI ALPHA 1 (COL11A1) IS A NOVEL STROMAL MEDIATOR OF CHEMORESISTANCE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-017.