Machine Learning Discoveries of WLS-X Synergy in ETC-1922159 Treated Colorectal Cancer Cells

Machine learning discoveries of WLS-X synergy in ETC-1922159 treated colorectal cancer cells shriprakash sinha Independent Researcher; Orcid ID : orcid.org/0000-0001-7027-5788 104-Madhurisha Heights Phase 1, Risali, Bhilai-490006, India Abstract Wntless (WLS) is a receptor required for WNT secretion. WLS is a cargo for the retromer complex. In the absence of retromer, WLS is degraded in lysosomes and the WNT secretion is impaired. In colorectal cancer (CRC) cells treated with ETC1922159, WLS was found to be up regulated along with other genes. A recently developed search engine ranked combinations of WLS-X (X, a particular gene/protein) at 2nd order level after drug administration. Some combinations have been experimentally validated, while many remain untested/unexplored. These rankings reveal which WLS-X combinations might be working synergistically in CRC. In this research work, I cover combinations of WLS with WNT, prolactin regulatory element-binding protein (SEC12), member RAS oncogene family (RAB), vacuolar protein sorting protein, a component of the retromer complex (VPS), sorting nexin (SNX), ADP ribosylation factors (ARF), ubiquitin conjugating enzyme E2 (UBE2), ATPases and transmembrane protein (TMEM) family. Keywords: WLS, Porcupine inhibitor ETC-1922159, Sensitivity analysis, Colorectal cancer. 1. Introduction 1.1. Wnt secretion Contrary to the signaling phenomena, the secretion phenomena is about the release and transportation of the WNT protein/ligand in and out of the cell, respectively. Briefly, the WNT proteins that are synthesized with the endoplasmic reticulum (ER), are known to5 be palmitoyleated via the Porcupine (PORCN) to form the WNT ligand, which is then ready for transportation Tanaka et al. [1]. It is believed that these ligands are then transported via the EVI/WNTLESS transmembrane complex out of the cell (Banziger et al. IML dicoveries of WLS-X synergy in ETC-1922159 treated CRC cells Email address: [email protected] (shriprakash sinha) 1Aspects of unpublished work were presented in a poster session at the first Wnt Gordon Research Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA. Preprint submitted to Elsevier December 31, 2024 [2], Bartscherer et al. [3] and Goodman et al. [4]). The EVI/WNTLESS themselves are known to reside in the Golgi bodies and interaction with the WNT ligands for the10 later’s glycosylation Kurayoshi et al. [5] & Gao and Hannoush [6]. Once outside the cell, the WNTs then interact with the cell receptors, as explained in the foregoing paragraph, to induce the Wnt signaling. Of importance is the fact that the EVI/WNTLESS also need a transporter in the from of a complex termed as Retromer. Voloshanenko et al. [7] show that WLS and WNT3 are highly expressed in colon15 carcinomas and EVI/WLS is required for high levels of WNT pathway activation. Further, Chua et al. [8] demonstrate that clinical osteosarcoma samples show high WLS and β-catenin expression. In colorectal cancer cells treated with ETC-1922159, WLS was found to be up regulated along with other genes. Some of the WLS-X (X, a particular gene/protein) combinations have been experimentally validated, while many remain20 untested/unexplored. I use the machine learning based search engine (the next section) to rank/prioritize these combinations to reveal untested/unexplored combinations. 1.2. Combinatorial search problem and a possible solution In a recently published work Sinha [9], a frame work of a search engine was developed which can rank combinations of factors (genes/proteins) in a signaling pathway.25 Readers are requested to go through the adaptation of the above mentioned work for gaining deeper insight into the working of the pipeline and its use of published data set generated after administration of ETC-1922159, Sinha [10]. The work uses SVM package by Joachims [11] in https://www.cs.cornell.edu/people/tj/svm_light/ svm_rank.html. I use the adaptation to rank 2nd order gene combinations.30 2. Results & Discussion 2.1. WLS related synergies 2.1.1. WLS-SEC Sun et al. [12] show that WLS forms a complex with SEC12. Binding of mature WNT to WLS increases WLS-SEC12 interaction and promotes association of WLS35 with SAR1 (a key activator of the COPII machinery). Mutant WLS that fail to communicate with the COPII machinery cannot effectively support WNT secretion. In colorectal cancer cells treated with ETC-1922159, SEC family and WLS, were found to be up regulated and recorded independently. I was able to rank 2nd order combination of SEC family and WLS, that were up regulated.40 Table 1 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 2 generated from analysis of the ranks in table 1. The table 1 shows rankings of SEC family w.r.t WLS. SEC24C - WLS shows low ranking of 1560 (laplace) and 622 (rbf). This low ranking points to the fact that the combination is not relevant after ETC-1922159 treatment of CRC, however, it might be prevalent in45 CRC, before treatment. Further, SEC24D - WLS show high ranking of 1644 (laplace), 2092 (linear) and 1679 (rbf). SEC31A - WLS show high ranking of 1856 (laplace) and 1793 (linear). 2 This high ranking points to the fact that the combination is relevant after ETC-1922159 treatment of CRC, however, it might not be prevalent in CRC, before treatment.50 RANKING SEC FAMILY VS WLS RANKING OF SEC FAMILY W.R.TWLS laplace linear rbf SEC24C - WLS 1560 2089 622 SEC24D - WLS 1644 2092 1679 SEC31A - WLS 1856 1793 831 Table 1: 2nd order interaction ranking between WLS VS SEC family members. One can also interpret the results of the table 1 graphically, with the following influences - •SEC family w.r.t WLS with WLS −>SEC-24C/24D/31A. UNEXPLORED COMBINATORIAL HYPOTHESES SEC family w.r.t WLS SEC-24C WLS (before ETC-1922159 treatment of CRC) SEC-24D/31A WLS (after ETC-1922159 treatment of CRC) Table 2: 2nd order combinatorial hypotheses between WLS and SEC family members. 2.1.2. WLS-RAB Das et al. [13] show that WNT secretion is dependent on RAB8A mediated transport of GPR177 (WNTLESS). GPR177 was found to bind with RAB8A, depletion55 of which compromised GPR177 traffic, thereby weakening the secretion of multiple WNTs. Further, Sun et al. [12] tabulate proteomic identification of RAB family in wild-type WLS and WLS1−491 immunoprecipitates. In colorectal cancer cells treated with ETC-1922159, RAB family and WLS, were found to be up regulated and recorded independently. I was able to rank 2nd order combination of RAB family and WLS, that60 were up regulated. Table 3 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 4 generated from analysis of the ranks in table 3. The table 3 shows rankings of RAB family w.r.t WLS. RAB24 - WLS shows low ranking of 249 (laplace), 739 (linear) and 1344 (rbf). RAB3B - WLS shows low ranking65 of 642 (laplace), 459 (linear) and 828 (rbf). RAB22A - WLS shows low ranking of 757 (laplace), 657 (linear) and 1078 (rbf). RAB5A - WLS shows low ranking of 771 3 (laplace) and 267 (rbf). RAB9A - WLS shows low ranking of 858 (laplace), 1562 (linear) and 371 (rbf). RAB4B - WLS shows low ranking of 1273 (laplace) and 572 (linear). RAB8A - WLS shows low ranking of 919 (linear) and 917 (rbf). RAB1A70 - WLS shows low ranking of 658 (linear) and 1535 (rbf). This low ranking points to the fact that the combination is not relevant after ETC-1922159 treatment of CRC, however, it might be prevalent in CRC, before treatment. Further, RAB25 - WLS show high ranking of 1939 (linear) and 2498 (rbf). RAB3GAP1 - WLS show high ranking of 2438 (linear) and 2103 (rbf). RAB11FIP1 - WLS show75 high ranking of 1561 (laplace) and 1653 (linear). RAB7A - WLS show high ranking of 1870 (laplace) and 2465 (rbf). RAB11A - WLS show high ranking of 2135 (laplace) and 2148 (linear). RAB1B - WLS show high ranking of 2237 (laplace), 2002 (linear) and 1730 (rbf). This high ranking points to the fact that the combination is relevant after ETC-1922159 treatment of CRC, however, it might not be prevalent in CRC, before80 treatment. RANKING RAB FAMILY VS WLS RANKING OF RAB FAMILY W.R.TWLS laplace linear rbf laplace linear rbf RAB24 - WLS 249 739 1344 RAB3B - WLS 642 459 828 RAB22A - WLS 757 657 1078 RAB5A - WLS 771 1688 267 RAB9A - WLS 858 1562 371 RAB4B - WLS 1273 572 2021 RAB25 - WLS 1445 1939 2498 RAB3GAP1 - WLS 1452 2438 2103 RAB11FIP1 - WLS 1561 1653 185 RAB8A - WLS 1751 919 917 RAB7A - WLS 1870 959 2465 RAB11A - WLS 2135 2148 1177 RAB1B - WLS 2237 2002 1730 RAB1A - WLS 2394 658 1535 Table 3: 2nd order interaction ranking between WLS VS RAB family members. One can also interpret the results of the table 3 graphically, with the following influences - •RAB family w.r.t WLS with WLS −>RAB-24/3B/22A/5A/9A/4B/8A/1A (before ETC-1922159 treatment of CRC) and WLS −>RAB-25/3GAP1/11FIP1/7A/11A/1B (after ETC-1922159 treatment of CRC).85 UNEXPLORED COMBINATORIAL HYPOTHESES RAB family w.r.t WLS RAB-24/3B/22A/5A/9A/4B/8A/1A WLS (before ETC-1922159 treatment of CRC) RAB-25/3GAP1/11FIP1/7A/11A/1B WLS (after ETC-1922159 treatment of CRC) Table 4: 2nd order combinatorial hypotheses between WLS and RAB family members. 2.1.3. WLS-VPS Belenkaya et al. [14] examined the role of VPS35 (a retromer subunit), in WNT signaling. They provide compelling evidence that VPS35 colocalizes in endosomes and interacts with WLS and WLS becomes unstable in the absence of retromer activity. 4 Further, Sun et al. [12] tabulate proteomic identification of VPS family in wild-type90 WLS and WLS1−491 immunoprecipitates. In colorectal cancer cells treated with ETC1922159, VPS family and WLS, were found to be up regulated and recorded independently. I was able to rank 2nd order combination of VPS family and WLS, that were up regulated. Table 5 shows rankings of these combinations. Followed by this is the unexplored95 combinatorial hypotheses in table 6 generated from analysis of the ranks in table 5. The table 5 shows rankings of VPS family w.r.t WLS. VPS37C - WLS shows low ranking of 1305 (laplace), 1381 (linear) and 1208 (rbf). VPS33B - WLS shows low ranking of 967 (linear) and 604 (rbf). This low ranking points to the fact that the combination is not relevant after ETC-1922159 treatment of CRC, however, it might be prevalent in100 CRC, before treatment. Further, VPS37B - WLS show high ranking of 1715 (laplace), 2182 (linear) and 1821 (rbf). VPS28 - WLS show high ranking of 2091 (laplace) and 2453 (rbf). VPS4B - WLS show high ranking of 2487 (laplace) and 1872 (rbf). This high ranking points to the fact that the combination is relevant after ETC-1922159 treatment of CRC, how-105 ever, it might not be prevalent in CRC, before treatment. RANKING VPS FAMILY VS WLS RANKING OF VPS FAMILY W.R.TWLS laplace linear rbf VPS37C - WLS 1305 1381 1208 VPS37B - WLS 1715 2182 1821 VPS33B - WLS 1777 967 604 VPS28 - WLS 2091 599 2453 VPS4B - WLS 2487 1028 1872 Table 5: 2nd order interaction ranking between WLS VS VPS family members. One can also interpret the results of the table 5 graphically, with the following influences - •VPS family w.r.t WLS with WLS −>VPS-37C/33B (before ETC-1922159 treatment of CRC) and WLS −>VPS-37B/28/4B (after ETC-1922159 treatment of CRC).110 2.1.4. WLS-SNX Harterink et al. [15] found that SNX3 has an evolutionarily conserved function in WLS recycling and WNT secretion. Brown et al. [16] show similar findings about SNX3 5 UNEXPLORED COMBINATORIAL HYPOTHESES VPS family w.r.t WLS VPS-37C/33B WLS (before ETC-1922159 treatment of CRC) VPS-37B/28/4B WLS (after ETC-1922159 treatment of CRC) Table 6: 2nd order combinatorial hypotheses between WLS and VPS family members. and WLS in mammalian neural tube closure. In colorectal cancer cells treated with ETC-1922159, SNX family and WLS, were found to be up regulated and recorded115 independently. I was able to rank 2nd order combination of SNX family and WLS, that were up regulated. Table 7 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 8 generated from analysis of the ranks in table 7. The table 7 shows rankings of SNX family w.r.t WLS. SNX9 - WLS shows low ranking of120 229 (laplace), 19 (linear) and 1501 (rbf). SNX11 - WLS shows low ranking of 1159 (laplace) and 1353 (rbf). This low ranking points to the fact that the combination is not relevant after ETC-1922159 treatment of CRC, however, it might be prevalent in CRC, before treatment. Further, SNX33 - WLS show high ranking of 1948 (laplace) and 2378 (linear).125 This high ranking points to the fact that the combination is relevant after ETC-1922159 treatment of CRC, however, it might not be prevalent in CRC, before treatment. RANKING SNX FAMILY VS WLS RANKING OF SNX FAMILY W.R.TWLS laplace linear rbf SNX9 - WLS 229 19 1501 SNX11 - WLS 1159 1691 1353 SNX33 - WLS 1948 2378 53 Table 7: 2nd order interaction ranking between WLS VS SNX family members. One can also interpret the results of the table 7 graphically, with the following influences - •SNX family w.r.t WLS with WLS −>SNX-9/11 (before ETC-1922159 treatment of CRC) and WLS −>SNX-33 (after ETC-1922159 treatment of CRC).130 2.1.5. WLS-ARF Yu et al. [17] show that WLS Golgi-to-ER retrieval requires the COPI regulator ARF 6 UNEXPLORED COMBINATORIAL HYPOTHESES SNX family w.r.t WLS SNX-9/11 WLS (before ETC-1922159 treatment of CRC) SNX-33 WLS (after ETC-1922159 treatment of CRC) Table 8: 2nd order combinatorial hypotheses between WLS and SNX family members. as well as ERGIC2. In colorectal cancer cells treated with ETC-1922159, ARF family and WLS, were found to be up regulated and recorded independently. I was able to rank 2nd order combination of ARF family and WLS, that were up regulated.135 Table 9 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 10 generated from analysis of the ranks in table 9. The table 9 shows rankings of ARF family w.r.t WLS. ARFGAP3 - WLS shows low ranking of 480 (laplace) and 1075 (rbf). ARF3 - WLS shows low ranking of 866 (laplace), 736 (linear) and 516 (rbf). ARF6 - WLS shows low ranking of 1291 (laplace)140 and 855 (rbf). ARF1 - WLS shows low ranking of 1526 (laplace) and 1170 (linear). This low ranking points to the fact that the combination is not relevant after ETC1922159 treatment of CRC, however, it might be prevalent in CRC, before treatment. Further, ARF4 - WLS shows high ranking of 2040 (laplace) and 2414 (linear). This high ranking points to the fact that the combination is relevant after ETC-1922159145 treatment of CRC, however, it might not be prevalent in CRC, before treatment. RANKING ARF FAMILY VS WLS RANKING OF ARF FAMILY W.R.TWLS laplace linear rbf ARFGAP3 - WLS 480 2111 1075 ARF3 - WLS 866 736 516 ARF6 - WLS 1291 1974 855 ARF1 - WLS 1526 1170 2079 ARF4 - WLS 2040 2414 1062 Table 9: 2nd order interaction ranking between WLS VS ARF family members. One can also interpret the results of the table 9 graphically, with the following influences - •ARF family w.r.t WLS with WLS −>ARF-GAP3/3/6/1 (before ETC1922159 treatment of CRC) and WLS −>ARF-4 (after ETC-1922159 treatment of 7 CRC).150 UNEXPLORED COMBINATORIAL HYPOTHESES ARF family w.r.t WLS ARF-GAP3/3/6/1 WLS (before ETC-1922159 treatment of CRC) ARF-4 WLS (after ETC-1922159 treatment of CRC) Table 10: 2nd order combinatorial hypotheses between WLS and ARF family members. 2.1.6. WLS-UBE2 Wolf et al. [18] found that EVI/WLS is ubiquitylated and degraded in cells irrespective of their level of WNT production. This ubiquitylation is mediated by the E2 ubiquitin-conjugating enzymes UBE2K, UBE2J2 and UBE2N. In colorectal cancer cells treated with ETC-1922159, UBE2 family and WLS, were found to be up regu-155 lated and recorded independently. I was able to rank 2nd order combination of UBE2 family and WLS, that were up regulated. Table 11 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 12 generated from analysis of the ranks in table 11. The table 11 shows rankings of UBE2 family w.r.t WLS. UBE2H - WLS shows160 low ranking of 868 (laplace), 1409 (linear) and 1051 (rbf). UBE2J1 - WLS shows low ranking of 957 (laplace) and 1346 (linear). UBE2F - WLS shows low ranking of 1379 (laplace), 356 (linear) and 221 (rbf). This low ranking points to the fact that the combination is not relevant after ETC-1922159 treatment of CRC, however, it might be prevalent in CRC, before treatment.165 Further, UBE2A - WLS shows high ranking of 1865 (laplace) and 2340 (linear). UBE2Z - WLS shows high ranking of 2032 (laplace) and 2265 (rbf). UBE2B - WLS shows high ranking of 2353 (laplace) and 1925 (rbf). This high ranking points to the fact that the combination is relevant after ETC-1922159 treatment of CRC, however, it might not be prevalent in CRC, before treatment.170 One can also interpret the results of the table 11 graphically, with the following influences - •UBE2 family w.r.t WLS with WLS −>UBE2-H/J1/F (before ETC1922159 treatment of CRC) and WLS −>UBE2-A/Z/B (after ETC-1922159 treatment of CRC). 2.1.7. WLS-ATPases175 McGough et al. [19] demonstrate the role of SNX3 for WNTLESS transport and report that SNX3 associates with a membrane remodelling complex composed of MON2, DOPEY2 and the putative aminophospholipid translocase, ATP9A. ATP9A comes under the category of one of the P-type ATPases (under the general category of ATPases). In colorectal cancer cells treated with ETC-1922159, ATP family and WLS, were found180 to be up regulated and recorded independently. I was able to rank 2nd order combination of ATP family and WLS, that were up regulated. 8 RANKING UBE2 FAMILY VS WLS RANKING OF UBE2 FAMILY W.R.TWLS laplace linear rbf UBE2H - WLS 868 1409 1051 UBE2J1 - WLS 957 1346 2165 UBE2F - WLS 1379 356 221 UBE2A - WLS 1865 2340 1213 UBE2Z - WLS 2032 1584 2265 UBE2B - WLS 2353 816 1925 Table 11: 2nd order interaction ranking between WLS VS UBE2 family members. UNEXPLORED COMBINATORIAL HYPOTHESES UBE2 family w.r.t WLS UBE2-H/J1/F WLS (before ETC-1922159 treatment of CRC) UBE2-A/Z/B WLS (after ETC-1922159 treatment of CRC) Table 12: 2nd order combinatorial hypotheses between WLS and UBE2 family members. Table 13 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 14 generated from analysis of the ranks in table 13. The table 13 shows rankings of ATP family w.r.t WLS. ATP1B1 - WLS shows185 low ranking of 302 (laplace) and 241 (rbf). ATP2B4 - WLS shows low ranking of 303 (laplace), 236 (linear) and 335 (rbf). ATP2A2 - WLS shows low ranking of 340 (laplace), 1116 (linear) and 76 (rbf). ATP13A2 - WLS shows low ranking of 657 (laplace), 306 (linear) and 1173 (rbf). ATP2B1 - WLS shows low ranking of 778 (laplace) and 346 (rbf). This low ranking points to the fact that the combination is not190 relevant after ETC-1922159 treatment of CRC, however, it might be prevalent in CRC, before treatment. Further, ATP6V1E1 - WLS shows high ranking of 1660 (laplace) and 1924 (rbf). ATP10B - WLS shows high ranking of 1810 (laplace) and 1527 (rbf). ATP6V1D - WLS shows high ranking of 1897 (laplace) and 1807 (linear). ATP11B - WLS shows high195 ranking of 1929 (laplace), 2336 (linear) and 2421 (rbf). ATP6V0D1 - WLS shows high ranking of 2435 (laplace), 1724 (linear) and 2289 (rbf). This high ranking points to the fact that the combination is relevant after ETC-1922159 treatment of CRC, however, it 9