Tumor microenvironment conditions that favor vessel co-option in colorectal cancer liver metastases: A theoretical model.

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.

Neutrophils expressing lysyl oxidase-like 4 protein are present in colorectal cancer liver metastases resistant to anti-angiogenic therapy.

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.

Intracellular Delivery of Nanomaterials via an Inertial Microfluidic Cell Hydroporator.

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.

A Retrospective Study on the Role of Metformin in Colorectal Cancer Liver Metastases.

Colorectal cancer liver metastases (CRCLMs) have two main histopathological growth patterns (HPGs): desmoplastic (DHGP) and replacement (RHGP). The vascularization in DHGP tumours is angiogenic, while the RHGP tumours exert vessel co-option vasculature. The presence of vessel co-option tumours is associated with poor response to anti-angiogenic agents and chemotherapy, as well as a worse prognosis. Metformin has been shown to influence the progression and vasculature of tumours in different cancers. However, its role in CRCLM is poorly understood. Herein, we conducted a retrospective cohort study to examine the role of metformin in CRCLM. A dataset of 108 patients was screened, of which 20 patients used metformin. The metformin user patients did not use metformin as an anticancer agent. We noticed a significantly lower percentage of CRCLM patients with vessel co-opting RHGP tumours in the population that used metformin compared to CRCLM patients who did not use metformin. Similar results were obtained when we compared the ratio of recurrence and extrahepatic metastases incidence. Moreover, the metformin user patients had significantly higher survival outcome compared to nonusers. Collectively, our data suggest that metformin administration is likely associated with better prognosis of CRCLM.

The molecular mechanisms underlying neutrophil infiltration in vessel co-opting colorectal cancer liver metastases.

Colorectal cancer liver metastases (CRCLMs) have two major histopathological growth patterns (HGPs): desmoplastic (DHGP) and replacement (RHGP). The DHGP tumours derive their vasculature by angiogenesis, while the RHGP tumours use vessel co-option. Various studies have associated RHGP tumours with an unfavourable prognosis, as well as high levels of resistance to anti-angiogenic agents and chemotherapy. Recently, we reported higher numbers of neutrophils in the tumour microenvironment (TME) of vessel co-opting tumours compared to their angiogenic counterparts. However, the molecular mechanisms underlying this phenotype are unclear. Herein, we suggested a positive correlation between the expression of angiopoietin-1 (Ang1) in the hepatocytes and the presence of neutrophils in vessel co-opting tumours. Importantly, upregulation of Ang1 in the hepatocytes is associated with the presence of runt-related transcription factor-1 (RUNX1) in the neighboring cancer cells in vitro and in vivo. Altogether, our data suggest the molecular mechanisms by which neutrophils are infiltrated in vessel co-opting CRCLM lesions. This finding may yield novel therapeutic strategies for CRCLM patients in future.

Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3.

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.

Cancer Cells Promote Phenotypic Alterations in Hepatocytes at the Edge of Cancer Cell Nests to Facilitate Vessel Co-Option Establishment in Colorectal Cancer Liver Metastases.

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.

Relevance of the Bruton Tyrosine Kinase as a Target for COVID-19 Therapy.

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.

Runt related transcription factor-1 plays a central role in vessel co-option of colorectal cancer liver metastases.

Colorectal cancer liver metastasis (CRCLM) has two major histopathological growth patterns: angiogenic desmoplastic and non-angiogenic replacement. The replacement lesions obtain their blood supply through vessel co-option, wherein the cancer cells hijack pre-existing blood vessels of the surrounding liver tissue. Consequentially, anti-angiogenic therapies are less efficacious in CRCLM patients with replacement lesions. However, the mechanisms which drive vessel co-option in the replacement lesions are unknown. Here, we show that Runt Related Transcription Factor-1 (RUNX1) overexpression in the cancer cells of the replacement lesions drives cancer cell motility via ARP2/3 to achieve vessel co-option. Furthermore, overexpression of RUNX1 in the cancer cells is mediated by Transforming Growth Factor Beta-1 (TGFß1) and thrombospondin 1 (TSP1). Importantly, RUNX1 knockdown impaired the metastatic capability of colorectal cancer cells in vivo and induced the development of angiogenic lesions in liver. Our results confirm that RUNX1 may be a potential target to overcome vessel co-option in CRCLM.

Inhibition of collagen XI alpha 1-induced fatty acid oxidation triggers apoptotic cell death in cisplatin-resistant ovarian cancer.

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 ß-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, α1ß1 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.

Amelioration of age-related brain function decline by Bruton's tyrosine kinase inhibition.

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.

Angiopoietin1 Deficiency in Hepatocytes Affects the Growth of Colorectal Cancer Liver Metastases (CRCLM).

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 chemonaïve 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).

BTK modulates p73 activity to induce apoptosis independently of p53.

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.

BTK: a two-faced effector in cancer and tumour suppression.

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.

Inhibitor of apoptosis proteins (IAPs) mediate collagen type XI alpha 1-driven cisplatin resistance in ovarian cancer.

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 α1ß1 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 α1ß1/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.

BTK blocks the inhibitory effects of MDM2 on p53 activity.

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.

BTK Modulates p53 Activity to Enhance Apoptotic and Senescent Responses.

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.