bims-pimaco 2021-07-04 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2021–07–04
fourteen papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Quadruple-editing of MAPK and PI3K pathways effectively blocks the progression of KRAS-mutated colorectal cancer cells.
Proteomic Profiling of BRAFV600E Mutant Colon Cancer Cells Reveals the Involvement of Nucleophosmin/c-Myc Axis in Modulating the Response and Resistance to BRAF Inhibition by Vemurafenib.
Insulin-Like Growth Factor 1 (IGF-1) Signaling in Glucose Metabolism in Colorectal Cancer.
Regulation of mTORC1 activity by the Golgi apparatus.
Ras Isoforms from Lab Benches to Lives-What Are We Missing and How Far Are We?
Colorectal Cancer Cells Ectopically Express Hepcidin to Sequester Iron.
Drugging the Undruggable: Advances on RAS Targeting in Cancer.
Chemical and Structural Strategies to Selectively Target mTOR Kinase.
Spotlight on Accessory Proteins: RTK-RAS-MAPK Modulators as New Therapeutic Targets.
The Ras dimer structure.
In vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphate.
AKT1 and PTEN show the highest affinities among phosphoinositide binding proteins for the second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2.
Fibrinogen activates focal adhesion kinase (FAK) promoting colorectal adenocarcinoma growth.
Establishment of a Novel In Vitro Model of Endometriosis with Oncogenic KRAS and PIK3CA Mutations for Understanding the Underlying Biology and Molecular Pathogenesis.

Cancer Sci. 2021 Jun 29.

Quadruple-editing of MAPK and PI3K pathways effectively blocks the progression of KRAS-mutated colorectal cancer cells.

Zaozao Wang, Bin Kang, Qianqian Gao, Lei Huang, Jiabo Di, Yingcong Fan, Jianhong Yu, Beihai Jiang, Feng Gao, Dan Wang, Haixi Sun, Ying Gu, Jian Li, Xiangqian Su.

  Mutated KRAS promotes the activation of MAPK pathway and the progression of colorectal cancer (CRC) cells. Aberrant activation of PI3K pathway strongly attenuates the efficacy of MAPK suppression in KRAS-mutated CRC. The development of a novel strategy targeting dual-pathway is therefore highly essential for the therapy of KRAS-mutated CRC. In this study, a quadruple-depleting system for KRAS, MEK1, PIK3CA, and MTOR genes based on CRISPR/SaCas9 was developed. Serotype 5 of adenovirus (ADV5) was integrated with two engineered proteins, an adaptor and a protector, to form ADV-protein complex (APC) for systemic delivery of the CRISPR system. The quadruple-editing could significantly inhibit MAPK and PI3K pathways in CRC cells with oncogenic mutations of KRAS and PIK3CA or with KRAS mutation and compensated PI3K activation. Compared with MEK and PI3K/MTOR inhibitors, the quadruple-editing induced more significant survival inhibition on primary CRC cells with oncogenic mutations of KRAS and PIK3CA. The adaptor specifically targeting EpCAM and the hexon-shielding protector could dramatically enhance ADV5 infection efficiency to CRC cells, and significantly reduce off-targeting tropisms to many organs except colon. Moreover, the quadruple-editing intravenously delivered by APC significantly blocked dual-pathway and tumor growth of KRAS-mutated CRC cells, without influencing normal tissues in cell- and patient-derived xenograft models. Therefore, APC-delivered quadruple-editing of MAPK and PI3K pathways shows a promising therapeutic potential for KRAS-mutated CRC.

Keywords:  ADV-protein complex; KRAS-mutated colorectal cancer; MAPK pathway; PI3K pathway; Quadruple-gene editing

DOI:  https://doi.org/10.1111/cas.15049
Int J Mol Sci. 2021 Jun 08. pii: 6174. [Epub ahead of print]22(12):

Proteomic Profiling of BRAFV600E Mutant Colon Cancer Cells Reveals the Involvement of Nucleophosmin/c-Myc Axis in Modulating the Response and Resistance to BRAF Inhibition by Vemurafenib.

Petra Grbčić, Dora Fučkar Čupić, Tania Gamberi, Sandra Kraljević Pavelić, Mirela Sedić.

  BRAFV600E mutations are found in approximately 10% of colorectal cancer patients and are associated with worse prognosis and poor outcomes with systemic therapies. The aim of this study was to identify novel druggable features of BRAFV600E-mutated colon cancer (CC) cells associated with the response and resistance to BRAFV600E inhibitor vemurafenib. Towards this aim, we carried out global proteomic profiling of BRAFV600E mutant vs. KRAS mutant/BRAF wild-type and double wild-type KRAS/BRAF CC cells followed by bioinformatics analyses. Validation of selected proteomic features was performed by immunohistochemistry and in silico using the TCGA database. We reveal an increased abundance and activity of nucleophosmin (NPM1) in BRAFV600E-mutated CC in vitro, in silico and in tumor tissues from colon adenocarcinoma patients and demonstrate the roles of NPM1 and its interaction partner c-Myc in conveying the resistance to vemurafenib. Pharmacological inhibition of NPM1 effectively restored the sensitivity of vemurafenib-resistant BRAF-mutated CC cells by down-regulating c-Myc expression and activity and consequently suppressing its transcriptional targets RanBP1 and phosphoserine phosphatase that regulate centrosome duplication and serine biosynthesis, respectively. Altogether, findings from this study suggest that the NPM1/c-Myc axis could represent a promising therapeutic target to thwart resistance to vemurafenib in BRAF-mutated CC.

Keywords:  BRAFV600E mutation; PLX4032; bioinformatics; c-Myc; chemoresistance; colorectal cancer; nucleophosmin; proteomic; vemurafenib

DOI:  https://doi.org/10.3390/ijms22126174
Int J Mol Sci. 2021 Jun 16. pii: 6434. [Epub ahead of print]22(12):

Insulin-Like Growth Factor 1 (IGF-1) Signaling in Glucose Metabolism in Colorectal Cancer.

Aldona Kasprzak.

  Colorectal cancer (CRC) is one of the most common aggressive carcinoma types worldwide, characterized by unfavorable curative effect and poor prognosis. Epidemiological data re-vealed that CRC risk is increased in patients with metabolic syndrome (MetS) and its serum components (e.g., hyperglycemia). High glycemic index diets, which chronically raise post-prandial blood glucose, may at least in part increase colon cancer risk via the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway. However, the underlying mechanisms linking IGF-1 and MetS are still poorly understood. Hyperactivated glucose uptake and aerobic glycolysis (the Warburg effect) are considered as a one of six hallmarks of cancer, including CRC. However, the role of insulin/IGF-1 signaling during the acquisition of the Warburg metabolic phenotypes by CRC cells is still poorly understood. It most likely results from the interaction of multiple processes, directly or indirectly regulated by IGF-1, such as activation of PI3K/Akt/mTORC, and Raf/MAPK signaling pathways, activation of glucose transporters (e.g., GLUT1), activation of key glycolytic enzymes (e.g., LDHA, LDH5, HK II, and PFKFB3), aberrant expression of the oncogenes (e.g., MYC, and KRAS) and/or overexpression of signaling proteins (e.g., HIF-1, TGF-β1, PI3K, ERK, Akt, and mTOR). This review describes the role of IGF-1 in glucose metabolism in physiology and colorectal carcinogenesis, including the role of the insulin/IGF system in the Warburg effect. Furthermore, current therapeutic strategies aimed at repairing impaired glucose metabolism in CRC are indicated.

Keywords:  Warburg effect; colorectal cancer; glucose metabolism; insulin-like growth factor 1 (IGF-1); metabolic risk factors; therapeutic strategies regulating insulin/IGF-1 signaling

DOI:  https://doi.org/10.3390/ijms22126434
Fac Rev. 2021 ;10 50

Regulation of mTORC1 activity by the Golgi apparatus.

Christian Makhoul, Paul A Gleeson.

  Mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) is a major signalling kinase in cells that regulates proliferation and metabolism and is controlled by extrinsic and intrinsic signals. The lysosome has received considerable attention as a major hub of mTORC1 activation. However, mTOR has also been located to a variety of other intracellular sites, indicating the possibility of spatial regulation of mTORC1 signalling within cells. In particular, there have been numerous recent reports of mTORC1 activation associated with the Golgi apparatus. Here, we review the evidence for the regulation of mTORC1 signalling at the Golgi in mammalian cells. mTORC1 signalling is closely linked to the morphology of the Golgi architecture; a number of Golgi membrane tethers/scaffolds that influence Golgi architecture in mammalian cells that directly or indirectly regulate mTORC1 activation have been identified. Perturbation of the Golgi mTORC1 pathway arising from fragmentation of the Golgi has been shown to promote oncogenesis. Here, we highlight the potential mechanisms for the activation mTORC1 at the Golgi, which is emerging as a major site for mTORC1 signalling.

Keywords:  Arf1; GAT4; GCC88; GOLPH3; Golgi architecture; Rab1A; actin; mTORC1; signalling; trans-Golgi network

DOI:  https://doi.org/10.12703/r/10-50
Int J Mol Sci. 2021 Jun 17. pii: 6508. [Epub ahead of print]22(12):

Ras Isoforms from Lab Benches to Lives-What Are We Missing and How Far Are We?

Arathi Nair, Katharina F Kubatzky, Bhaskar Saha.

  The central protein in the oncogenic circuitry is the Ras GTPase that has been under intense scrutiny for the last four decades. From its discovery as a viral oncogene and its non-oncogenic contribution to crucial cellular functioning, an elaborate genetic, structural, and functional map of Ras is being created for its therapeutic targeting. Despite decades of research, there still exist lacunae in our understanding of Ras. The complexity of the Ras functioning is further exemplified by the fact that the three canonical Ras genes encode for four protein isoforms (H-Ras, K-Ras4A, K-Ras4B, and N-Ras). Contrary to the initial assessment that the H-, K-, and N-Ras isoforms are functionally similar, emerging data are uncovering crucial differences between them. These Ras isoforms exhibit not only cell-type and context-dependent functions but also activator and effector specificities on activation by the same receptor. Preferential localization of H-, K-, and N-Ras in different microdomains of the plasma membrane and cellular organelles like Golgi, endoplasmic reticulum, mitochondria, and endosome adds a new dimension to isoform-specific signaling and diverse functions. Herein, we review isoform-specific properties of Ras GTPase and highlight the importance of considering these towards generating effective isoform-specific therapies in the future.

Keywords:  H-Ras; K-Ras; N-Ras; Ras; Ras-isoforms; cancer; oncogene; signaling; therapy

DOI:  https://doi.org/10.3390/ijms22126508
Cancer Discov. 2021 Jul 02.

Colorectal Cancer Cells Ectopically Express Hepcidin to Sequester Iron.

Colorectal cancer cells express hepcidin to accumulate iron, promoting nucleotide metabolism.

DOI: https://doi.org/10.1158/2159-8290.CD-RW2021-092
Genes (Basel). 2021 Jun 10. pii: 899. [Epub ahead of print]12(6):

Drugging the Undruggable: Advances on RAS Targeting in Cancer.

Miriam Molina-Arcas, Amit Samani, Julian Downward.

  Around 20% of all malignancies harbour activating mutations in RAS isoforms. Despite this, there is a deficiency of RAS-targeting agents licensed for therapeutic use. The picomolar affinity of RAS for GTP, and the lack of suitable pockets for high-affinity small-molecule binding, precluded effective therapies despite decades of research. Recently, characterisation of the biochemical properties of KRAS-G12C along with discovery of its 'switch-II pocket' have allowed development of effective mutant-specific inhibitors. Currently seven KRAS-G12C inhibitors are in clinical trials and sotorasib has become the first one to be granted FDA approval. Here, we discuss historical efforts to target RAS directly and approaches to target RAS effector signalling, including combinations that overcome limitations of single-agent targeting. We also review pre-clinical and clinical evidence for the efficacy of KRAS-G12C inhibitor monotherapy followed by an illustration of combination therapies designed to overcome primary resistance and extend durability of response. Finally, we briefly discuss novel approaches to targeting non-G12C mutant isoforms.

Keywords:  KRAS-G12C; RAS; cancer; oncogene; targeted therapy

DOI:  https://doi.org/10.3390/genes12060899
ChemMedChem. 2021 Jun 11.

Chemical and Structural Strategies to Selectively Target mTOR Kinase.

Chiara Borsari, Martina De Pascale, Matthias P Wymann.

  Dysregulation of the mechanistic target of rapamycin (mTOR) pathway is implicated in cancer and neurological disorder, which identifies mTOR inhibition as promising strategy for the treatment of a variety of human disorders. First-generation mTOR inhibitors include rapamycin and its analogues (rapalogs) which act as allosteric inhibitors of TORC1. Structurally unrelated, ATP-competitive inhibitors that directly target the mTOR catalytic site inhibit both TORC1 and TORC2. Here, we review investigations of chemical scaffolds explored for the development of highly selective ATP-competitive mTOR kinase inhibitors (TORKi). Extensive medicinal chemistry campaigns allowed to overcome challenges related to structural similarity between mTOR and the phosphoinositide 3-kinase (PI3K) family. A broad region of chemical space is covered by TORKi. Here, the investigation of chemical substitutions and physicochemical properties has shed light on the compounds' ability to cross the blood brain barrier (BBB). This work provides insights supporting the optimization of TORKi for the treatment of cancer and central nervous system disorders.

Keywords:  BBB permeability; binding modes; chemical scaffolds; mTOR inhibitors; physicochemical properties

DOI:  https://doi.org/10.1002/cmdc.202100332
Biomolecules. 2021 Jun 16. pii: 895. [Epub ahead of print]11(6):

Spotlight on Accessory Proteins: RTK-RAS-MAPK Modulators as New Therapeutic Targets.

Silke Pudewell, Mohammad Reza Ahmadian.

  The RTK-RAS-MAPK axis is one of the most extensively studied signaling cascades and is related to the development of both cancers and RASopathies. In the last 30 years, many ideas and approaches have emerged for directly targeting constituent members of this cascade, predominantly in the context of cancer treatment. These approaches are still insufficient due to undesirable drug toxicity, resistance, and low efficacy. Significant advances have been made in understanding the spatiotemporal features of the constituent members of the RTK-RAS-MAPK axis, which are linked and modulated by many accessory proteins. Given that the majority of such modulators are now emerging as attractive therapeutic targets, a very small number of accessory inhibitors have yet to be discovered.

Keywords:  ERK; KRAS; MEK; RAF kinase; RTK; adaptor proteins; anchoring proteins; docking proteins; scaffold proteins

DOI:  https://doi.org/10.3390/biom11060895
Chem Sci. 2021 May 04. 12(23): 8178-8189

The Ras dimer structure.

Till Rudack, Christian Teuber, Marvin Scherlo, Jörn Güldenhaupt, Jonas Schartner, Mathias Lübben, Johann Klare, Klaus Gerwert, Carsten Kötting.

Oncogenic mutated Ras is a key player in cancer, but despite intense and expensive approaches its catalytic center seems undruggable. The Ras dimer interface is a possible alternative drug target. Dimerization at the membrane affects cell growth signal transduction. In vivo studies indicate that preventing dimerization of oncogenic mutated Ras inhibits uncontrolled cell growth. Conventional computational drug-screening approaches require a precise atomic dimer model as input to successfully access drug candidates. However, the proposed dimer structural models are controversial. Here, we provide a clear-cut experimentally validated N-Ras dimer structural model. We incorporated unnatural amino acids into Ras to enable the binding of labels at multiple positions via click chemistry. This labeling allowed the determination of multiple distances of the membrane-bound Ras-dimer measured by fluorescence and electron paramagnetic resonance spectroscopy. In combination with protein-protein docking and biomolecular simulations, we identified key residues for dimerization. Site-directed mutations of these residues prevent dimer formation in our experiments, proving our dimer model to be correct. The presented dimer structure enables computational drug-screening studies exploiting the Ras dimer interface as an alternative drug target.

DOI: https://doi.org/10.1039/d1sc00957e
J Biol Chem. 2021 Jun 25. pii: S0021-9258(21)00719-5. [Epub ahead of print] 100919

In vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphate.

Daniel Pokorny, Linda Truebestein, Kaelin D Fleming, John E Burke, Thomas A Leonard.

  Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen-deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation, and that the PI3P binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

Keywords:  CISK; Sgk3; allosteric regulation; endosome; hydrogen-deuterium exchange mass spectrometry (HDX-MS); phosphatidylinositide 3-kinase (PI 3-kinase); phospholipid; serine/threonine protein kinase

DOI:  https://doi.org/10.1016/j.jbc.2021.100919
Biochem Biophys Res Commun. 2021 Jun 29. pii: S0006-291X(21)00948-7. [Epub ahead of print]568 110-115

AKT1 and PTEN show the highest affinities among phosphoinositide binding proteins for the second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2.

Nina Nelson, Adelia Razeto, Alessia Gilardi, Mira Grättinger, Johannes Kirchmair, Manfred Jücker.

  The phosphoinositides phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2] function as second messengers and have been implicated in cancerogenesis. The signalling events downstream of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are mediated through a complex network of phosphoinositide binding effector proteins and phosphatases. In this study, we compared the phosphoinositide effector proteins AKT1, TAPP1, TAPP2, VAV1 and P-REX1 and the phosphoinositide phosphatases PTEN, SHIP1 and INPP4B for their binding affinities to PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2 using Surface Plasmon Resonance. Our results demonstrate that all measured proteins except P-REX1 and VAV1 showed high affinity phosphoinositide binding with KD values in the nM to sub-nM range. Within the effector proteins, AKT1 showed the highest affinity for both PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Of the phosphoinositide phosphatases PTEN displayed the highest affinity towards PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The SHIP1 mutant E452K detected in carcinoma patients had a 100-fold increased affinity to PtdIns(3,4)P2 but not to PtdIns(3,4,5)P3 compared to SHIP1 WT. Distinct mutations in phosphoinositide binding proteins like the patient-derived SHIP1E452K mutant may be involved in the upregulation of PI(3,4)P2 -mediated signalling in tumor cells due to phosphoinositide trapping. Our results add further information to the complex hierarchy of phosphoinositide binding proteins helping to elucidate their functional role in cellular signal transduction.

Keywords:  Phosphatidylinositol-3,4,5-trisphosphate; Phosphatidylinositol-3,4-bisphosphate; Second messenger; Surface plasmon resonance

DOI:  https://doi.org/10.1016/j.bbrc.2021.06.027
J Thromb Haemost. 2021 Jun 30.

Fibrinogen activates focal adhesion kinase (FAK) promoting colorectal adenocarcinoma growth.

Bal Krishan Sharma, Duaa Mureb, Sumit Murab, Leah Rosenfeldt, Brenton Francisco, Rachel Cantrell, Rebekah Karns, Lindsey Romick-Rosendale, Miki Watanabe-Chailland, Jacob Mast, Matthew J Flick, Patrick W Whitlock, Joseph S Palumbo.

   BACKGROUND: We previously showed that fibrinogen is a major determinant of the growth of a murine model of colorectal cancer (CRC).
OBJECTIVE: Our aim was to define the mechanisms coupling fibrin(ogen) to CRC growth.
RESULTS: CRC tumors transplanted into the dorsal subcutis of Fib- mice were less proliferative and demonstrated increased senescence relative to those grown in Fib+ mice. RNA-Seq analyses of Fib+ and Fib- tumors revealed 213 differentially regulated genes. One gene highly upregulated in tumors from Fib- mice was stratifin, encoding 14-3-3σ, a master regulator of proliferation/senescence. In a separate cohort, we observed significantly increased protein levels of 14-3-3σ and its upstream and downstream targets (i.e., p53 and p21) in tumors from Fib- mice. In vitro analyses demonstrated increased tumor cell proliferation in a fibrin printed 3D environment compared to controls, suggesting that fibrin(ogen) in the tumor microenvironment promotes tumor growth in this context via a tumor cell intrinsic mechanism. In vivo analyses showed diminished activation of focal adhesion kinase (FAK), a key negative regulator of p53, in Fib- tumors. Furthermore, NMR-based metabolomics demonstrated significantly reduced metabolic activity in tumors from Fib- relative to Fib+ mice. Together these findings suggest that fibrin(ogen)-mediated engagement of colon cancer cells activates FAK, which inhibits p53 and its downstream targets including 14-3-3σ and p21, thereby promoting cellular proliferation and preventing senescence.
CONCLUSIONS: These studies suggest that fibrin(ogen) is an important component of the colon cancer microenvironment and may be exploited as a potential therapeutic target.

Keywords:  14-3-3σ; Fibrinogen; Focal adhesion kinase(FAK); cell senescence; colorectal adenocarcinoma; p53

DOI:  https://doi.org/10.1111/jth.15440
Cancers (Basel). 2021 Jun 25. pii: 3174. [Epub ahead of print]13(13):

Establishment of a Novel In Vitro Model of Endometriosis with Oncogenic KRAS and PIK3CA Mutations for Understanding the Underlying Biology and Molecular Pathogenesis.

Mohammad Mahmud Hossain, Kentaro Nakayama, Kamrunnahar Shanta, Sultana Razia, Masako Ishikawa, Tomoka Ishibashi, Hitomi Yamashita, Seiya Sato, Kouji Iida, Kosuke Kanno, Noriyoshi Ishikawa, Tohru Kiyono, Satoru Kyo.

  Endometriosis-harboring cancer-associated somatic mutations of PIK3CA and KRAS provides new opportunities for studying the multistep processes responsible for the functional and molecular changes in this disease. We aimed to establish a novel in vitro endometriosis model to clarify the functional behavior and molecular pathogenesis of this disorder. Immortalized HMOsisEC10 human ovarian endometriotic epithelial cell line was used in which KRAS and PIK3CA mutations were introduced. Migration, invasion, proliferation, and microarray analyses were performed using KRAS and PIK3CA mutant cell lines. In vitro assays showed that migration, invasion, and proliferation were significantly increased in KRAS and PIK3CA mutant cell lines, indicating that these mutations played causative roles in the aggressive behavior of endometriosis. Microarray analysis identified a cluster of gene signatures; among them, two significantly upregulated cancer-related genes, lysyl oxidase (LOX) and pentraxin3 (PTX3), were associated with cell proliferation, invasion, and migration capabilities. Furthermore, siRNA knockdown of the two genes markedly reduced the metastatic ability of the cells. These results suggest that endometriosis with KRAS or PIK3CA mutations can significantly enhance cell migration, invasion, and proliferation by upregulating LOX and PTX3. We propose that LOX and PTX3 silencing using small molecules could be an alternative therapeutic regimen for severe endometriosis.

Keywords:  KRAS; LOX; PIK3CA; PTX3; cell proliferation; epithelial cell line; invasion; microarray analysis; migration; ovarian endometriosis

DOI:  https://doi.org/10.3390/cancers13133174