Machine Learning Discoveries of STEAP3-X Synergy in etc-1922159 Treated Colorectal Cancer Cells

Machine learning discoveries of STEAP3-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 Six-transmembrane epithelial antigen of prostate 3 (STEAP3) is a metalloreductase that is capable of converting iron from an insoluble ferric (Fe3+) to a soluble ferrous (Fe2+) form. It has been found to be overexpressed in colorectal cancer. In colorectal cancer cells treated with ETC-1922159, it was found to be down-regulated. A recently developed search engine ranked combinations of STEAP3-X (X, a particular gene/protein) at 2nd order level after drug administration. In this research work, I cover combinations of STEAP3 with cyclin dependent kinase inhibitor (CDKN), glutathione peroxidase (GPX), solute carrier family (SLC), methyltransferase N6 adenosine methyltransferase complex catalytic subunit (METTL), b-cell leukemia / lymphoma protein (BCL), interleukin (IL) and transferrin receptor protein (TFR) family. Keywords: STEAP3, Porcupine inhibitor ETC-1922159, Sensitivity analysis, Colorectal cancer. 1. Introduction 1.1. Six-transmembrane epithelial antigen of prostate (STEAP) Sendamarai et al. [1] discuss the role of STEAP3 as a metalloreductase. They state that erythroid precursor cells uptake iron (Fe3+) by loading it to transferrin (TF) which then binds to the transferrin receptor (TFR) at the cell surface. This TF-TFR complex5 then enters the endosome. Upon endosomal acidification, iron is released from TF, reduced to Fe2+by STEAP3, and transported across the endosomal membrane by a divalent metal iron transporter. Ohgami et al. [2] further characterize and demonstrate that STEAP2, STEAP3, and STEAP4 not only reduce iron but also copper. Zhou et al. [3] and Lv et al. [4] show that STEAP3 promotes colorectal cancer. In colorectal10 cancer cells treated with ETC-1922159, STEAP3 was found to be down regulated, IML dicoveries of STEAP3-X synergy in ETC-1922159 treated CRC cells Email address: [email protected] (shriprakash sinha) 1Aspects of unpublished work were presented in a poster session at (1) the recently concluded first ever Wnt Gordon Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA. Preprint submitted to Preprint December 24, 2024 along with other genes. I present here machine learning based discoveries of STEAP3X combinations that might be working synergistically in colorectal cancer. Some of these combinations have already been tested in wet lab, however many others remain unexplored/untested. To address this, I use the following search engine, in the next15 section. 1.2. Combinatorial search problem and a possible solution In a recently published work Sinha [5], a frame work of a search engine was developed which can rank combinations of factors (genes/proteins) in a signaling pathway. Readers are requested to go through the adaptation of the above mentioned work for gaining20 deeper insight into the working of the pipeline and its use of published data set generated after administration of ETC-1922159, Sinha [6]. The work uses SVM package by Joachims [7] in https://www.cs.cornell.edu/people/tj/svm_light/svm_ rank.html. I use the adaptation to rank 2nd order gene combinations. 2. Results & Discussion25 2.1. STEAP3 related synergies 2.1.1. STEAP3-CDKN family In colon cancer, Na et al. [8] STEAP3 overexpression upregulated the expression of CDKN1C. In colorectal cancer cells treated with ETC-1922159, some of the family members of CDKN were down regulated, along with STEAP3. Individual recordings30 of these down regulations have been documented. I was able to rank 2nd order combination of CDKN family members with STEAP3, that were down regulated. 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 CDKN family w.r.t STEAP3. CDKN2C - STEAP335 shows low ranking of 906 (laplace) and 118 (linear). CDKN3 - STEAP3 shows low ranking of 1369 (laplace) and 223 (linear). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. CDKN2AIPNL did not show synergistic down regulation with STEAP3. One can also interpret the results of the table 1 graphically, with the following40 influences - •CDKN family w.r.t STEAP3 with STEAP3 −>CDKN-2C/3. 2.1.2. STEAP3-GPX family In renal cell carcinoma, Ye et al. [9] show that in STEAP3 knockdown group, expression of othe ferroptosis-related protein GPX4 was decreased. Similarly, Han et al. [10] show that knockdown of STEAP3 decreased the expression of GPX4 in ovarian cancer45 cells. In colorectal cancer cells treated with ETC-1922159, some of the family members of GPX were down regulated, along with STEAP3. Individual recordings of these down regulations have been documented. I was able to rank 2nd order combination of GPX family members with STEAP3, that were down regulated. 2 RANKING CDKN FAMILY VS STEAP3 RANKING OF CDKN FAMILY W.R.TSTEAP3 laplace linear rbf CDKN2C - STEAP3 906 118 2200 CDKN3 - STEAP3 1369 223 2556 CDKN2AIPNL - STEAP3 2353 1642 1362 Table 1: 2nd order interaction ranking between STEAP3 VS CDKN family members. UNEXPLORED COMBINATORIAL HYPOTHESES CDKN family w.r.t STEAP3 CDKN-2C/3 STEAP3 Table 2: 2nd order combinatorial hypotheses between STEAP3 and CDKN family members. Table 3 shows rankings of these combinations. Followed by this is the unexplored50 combinatorial hypotheses in table 4 generated from analysis of the ranks in table 3. The table 3 shows rankings of GPX family w.r.t STEAP3. GPX1 - STEAP3 shows low ranking of 333 (linear) and 812 (rbf). GPX1P1 - STEAP3 shows low ranking of 1064 (linear) and 1610 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment.55 RANKING GPX FAMILY VS STEAP3 RANKING OF GPX FAMILY W.R.TSTEAP3 laplace linear rbf GPX1 - STEAP3 2124 333 812 GPX1P1 - STEAP3 2328 1064 1610 Table 3: 2nd order interaction ranking between STEAP3 VS GPX family members. One can also interpret the results of the table 3 graphically, with the following influences - •GPX family w.r.t STEAP3 with STEAP3 −>GPX-1/1P1. 3 UNEXPLORED COMBINATORIAL HYPOTHESES GPX family w.r.t STEAP3 GPX-1/1P1 STEAP3 Table 4: 2nd order combinatorial hypotheses between STEAP3 and GPX family members. 2.1.3. STEAP3-SLC family Han et al. [10] show that knockdown of STEAP3 decreased the expression of SLC7A11 in ovarian cancer cells. In colorectal cancer cells treated with ETC-1922159, some of60 the family members of SLC were down regulated, along with STEAP3. Individual recordings of these down regulations have been documented. I was able to rank 2nd order combination of SLC family members with STEAP3, that were down regulated. Table 5 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 6 generated from analysis of the ranks in table 5.65 The table 5 shows rankings of SLC family w.r.t STEAP3. SLC19A3 - STEAP3 shows low ranking of 3 (laplace) and 58 (rbf). SLC25A19 - STEAP3 shows low ranking of 56 (laplace) and 600 (rbf). SLC39A10 - STEAP3 shows low ranking of 79 (laplace) and 292 (rbf). SLC12A8 - STEAP3 shows low ranking of 104 (laplace) and 846 (rbf). SLC25A35 - STEAP3 shows low ranking of 259 (laplace), 651 (linear) and70 728 (rbf). SLC28A3 - STEAP3 shows low ranking of 272 (laplace), 1113 (linear) and 458 (rbf). SLC7A8 - STEAP3 shows low ranking of 368 (laplace), 559 (linear) and 887 (rbf). SLC19A1 - STEAP3 shows low ranking of 429 (laplace) and 1395 (linear). SLC25A26 - STEAP3 shows low ranking of 548 (laplace), 230 (linear) and 982 (rbf). SLC16A1.AS1 - STEAP3 shows low ranking of 690 (laplace), 1232 (linear) and 54175 (rbf). SLC25A27 - STEAP3 shows low ranking of 1105 (laplace), 380 (linear) and 1569 (rbf). SLC35G1 - STEAP3 shows low ranking of 1108 (laplace) and 1302 (rbf). SLC39A8 - STEAP3 shows low ranking of 1175 (laplace) and 1564 (linear). SLC17A9 - STEAP3 shows low ranking of 1319 (laplace), 777 (linear) and 191 (rbf). SLC43A3 - STEAP3 shows low ranking of 1336 (laplace) and 341 (linear). SLC35E3 - STEAP380 shows low ranking of 922 (linear) and 690 (rbf). SLC12A2 - STEAP3 shows low ranking of 150 (linear) and 1223 (rbf). SLC25A32 - STEAP3 shows low ranking of 1203 (linear) and 1426 (rbf). SLC38A5 - STEAP3 shows low ranking of 1151 (linear) and 1343 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment.85 On the other hand, SLC4A7, SLC1A4, SLC18B1, SLC25A38, SLC28A2, SLC41A1, SLC25A15, SLC5A6, SLC35F2, SLC7A2, SLC43A1, SLC2A11, SLC25A14, SLC26A2, SLC25A40 and SLC6A6 do not show synergy with STEAP3 while synergistic down regulation. One can also interpret the results of the table 5 graphically, with the following90 influences - •SLC family w.r.t STEAP3 with STEAP3 −>SLC-19A3 / 25A19 / 39A10 / 12A8 / 25A35 / 28A3 / 7A8 / 19A1 / 25A26 / 16A1.AS1 / 25A27 / 35G1 / 39A8 / 17A9 / 43A3 / 35E3 / 12A2 / 25A32 / 38A5. 4 RANKING SLC FAMILY VS STEAP3 RANKING OF SLC FAMILY W.R.TSTEAP3 laplace linear rbf laplace linear rbf SLC19A3 - STEAP3 3 2632 58 SLC25A19 - STEAP3 56 2618 600 SLC39A10 - STEAP3 79 1648 292 SLC12A8 - STEAP3 104 2463 846 SLC25A35 - STEAP3 259 651 728 SLC28A3 - STEAP3 272 1113 458 SLC7A8 - STEAP3 368 559 887 SLC4A7 - STEAP3 417 2258 2277 SLC19A1 - STEAP3 429 1395 2288 SLC25A26 - STEAP3 548 230 982 SLC1A4 - STEAP3 654 1931 1557 SLC16A1.AS1 - STEAP3 690 1232 541 SLC25A27 - STEAP3 1105 380 1569 SLC35G1 - STEAP3 1108 1654 1302 SLC39A8 - STEAP3 1175 1564 1803 SLC18B1 - STEAP3 1318 1670 1853 SLC17A9 - STEAP3 1319 777 191 SLC43A3 - STEAP3 1336 341 1777 SLC25A38 - STEAP3 1519 1768 1697 SLC28A2 - STEAP3 1573 2082 931 SLC35E3 - STEAP3 1589 922 690 SLC41A1 - STEAP3 1591 2516 1965 SLC25A15 - STEAP3 1623 1703 624 SLC5A6 - STEAP3 1671 1866 1668 SLC35F2 - STEAP3 1734 107 1842 SLC43A1 - STEAP3 1832 431 2423 SLC7A2 - STEAP3 1942 302 2648 SLC12A2 - STEAP3 1991 150 1223 SLC25A32 - STEAP3 2063 1203 1426 SLC2A11 - STEAP3 2101 1991 1404 SLC25A14 - STEAP3 2120 308 2254 SLC26A2 - STEAP3 2240 1658 964 SLC25A40 - STEAP3 2269 1371 2206 SLC6A6 - STEAP3 2576 1519 2482 SLC38A5 - STEAP3 2682 1151 1343 Table 5: 2nd order interaction ranking between STEAP3 VS SLC family members. UNEXPLORED COMBINATORIAL HYPOTHESES SLC family w.r.t STEAP3 SLC-19A3/25A19/39A10/12A8/25A35/28A3/7A8/19A1 STEAP3 SLC-25A26/16A1.AS1/25A27/35G1/39A8/17A9/43A3 STEAP3 SLC-35E3/12A2/25A32/38A5 STEAP3 Table 6: 2nd order combinatorial hypotheses between STEAP3 and SLC family members. 2.1.4. STEAP3-METTL family In colorectal cancer, Zhou et al. [3] determined the related regulators for STEAP395 m6A modification, by knocking down m6A writers (METTL3, METTL14) and m6A readers (YTHDF1, YTHDF2). Their experiments shows that the protein level and mRNA level of STEAP3 were decreased by METTL14 and YTHDF2. In colorectal cancer cells treated with ETC-1922159, some of the family members of METTL were down regulated, along with STEAP3. Individual recordings of these down regulations100 have been documented. I was able to rank 2nd order combination of METTL family members with STEAP3, that were down 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 METTL family w.r.t STEAP3. METTL3 - STEAP3105 shows low ranking of 156 (laplace) and 326 (rbf). METTL16 - STEAP3 shows low ranking of 799 (laplace) and 654 (rbf). METTL12 - STEAP3 shows low ranking of 884 5 (laplace), 1147 (linear) and 687 (rbf). METTL21B - STEAP3 shows low ranking of 1228 (laplace) and 995 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. However,110 METTL1, METTL8, METTL13, METTL5, METTL21A, METTL17 and METTL2B did not show any down regulation synergy with STEAP3. RANKING METTL FAMILY VS STEAP3 RANKING OF METTL FAMILY W.R.TSTEAP3 laplace linear rbf METTL3 - STEAP3 156 1958 326 METTL16 - STEAP3 799 2196 654 METTL12 - STEAP3 884 1147 687 METTL21B - STEAP3 1228 2012 995 METTL1 - STEAP3 1579 1553 689 METTL8 - STEAP3 1659 2592 1638 METTL13 - STEAP3 1721 2335 1346 METTL5 - STEAP3 1960 1544 2485 METTL21A - STEAP3 2154 1937 1446 METTL17 - STEAP3 2239 1126 2370 METTL2B - STEAP3 2689 74 2242 Table 7: 2nd order interaction ranking between STEAP3 VS METTL family members. One can also interpret the results of the table 7 graphically, with the following influences - •METTL family w.r.t STEAP3 with STEAP3 −>METTL-3/16/12/21B. UNEXPLORED COMBINATORIAL HYPOTHESES METTL family w.r.t STEAP3 METTL-3/16/12/21B STEAP3 Table 8: 2nd order combinatorial hypotheses between STEAP3 and METTL family members. 6 2.1.5. STEAP3-BCL family115 In hepatocellular carcinoma, citetWang:2021steap3 STEAP3 promotes cancer cell proliferation. They found that anti-apoptotic protein BCL2 was substantially transcriptionally upregulated by STEAP3. In colorectal cancer cells treated with ETC-1922159, some of the family members of BCL were down regulated, along with STEAP3. Individual recordings of these down regulations have been documented. I was able to120 rank 2nd order combination of BCL family members with STEAP3, that were down regulated. 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 BCL family w.r.t STEAP3. BCL6B - STEAP3 shows125 low ranking of 65 (laplace) and 100 (rbf). BCL11A - STEAP3 shows low ranking of 854 (laplace) and 1322 (rbf). BCL11B - STEAP3 shows low ranking of 1026 (laplace) and 691 (rbf). BCL2L12 - STEAP3 shows low ranking of 1035 (laplace) and 1292 (linear). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. However, BCL9 and BCL7A130 did not show any down regulation synergy with STEAP3. RANKING BCL FAMILY VS STEAP3 RANKING OF BCL FAMILY W.R.TSTEAP3 laplace linear rbf BCL6B - STEAP3 65 2708 100 BCL11A - STEAP3 854 1793 1322 BCL11B - STEAP3 1026 2624 691 BCL2L12 - STEAP3 1035 1292 2235 BCL9 - STEAP3 2265 558 1963 BCL7A - STEAP3 2597 739 2309 Table 9: 2nd order interaction ranking between STEAP3 VS BCL family members. One can also interpret the results of the table 9 graphically, with the following influences - •BCL family w.r.t STEAP3 with STEAP3 −>BCL-6B/11A/11B/2L12. 2.1.6. STEAP3-IL family In hepatocellular carcinoma, Wang et al. [11] STEAP3 promotes cancer cell prolifer-135 ation. They found that inflammatory factors like IL-8 and IL-18 were substantially transcriptionally upregulated by STEAP3. In colorectal cancer cells treated with ETC7 UNEXPLORED COMBINATORIAL HYPOTHESES BCL family w.r.t STEAP3 BCL-6B/11A/11B/2L12 STEAP3 Table 10: 2nd order combinatorial hypotheses between STEAP3 and BCL family members. 1922159, some of the family members of IL were down regulated, along with STEAP3. Individual recordings of these down regulations have been documented. I was able to rank 2nd order combination of IL family members with STEAP3, that were down reg-140 ulated. 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 IL family w.r.t STEAP3. ILF3 - STEAP3 shows low ranking of 121 (laplace) and 926 (rbf). IL17RB - STEAP3 shows low ranking of 208145 (laplace) and 404 (rbf). IL17D - STEAP3 shows low ranking of 596 (laplace) and 705 (linear). IL33 - STEAP3 shows low ranking of 1070 (laplace) and 57 (linear). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. However, IL1RL2, ILF2 and IL17RD did not show any down regulation synergy with STEAP3.150 RANKING IL FAMILY VS STEAP3 RANKING OF IL FAMILY W.R.TSTEAP3 laplace linear rbf ILF3 - STEAP3 121 2314 926 IL17RB - STEAP3 208 2462 404 IL17D - STEAP3 596 705 2273 IL1RL2 - STEAP3 835 2234 1733 IL33 - STEAP3 1070 57 2098 ILF2 - STEAP3 1986 1029 2474 IL17RD - STEAP3 2352 589 2233 Table 11: 2nd order interaction ranking between STEAP3 VS IL family members. One can also interpret the results of the table 11 graphically, with the following 8 influences - •IL family w.r.t STEAP3 with STEAP3 −>IL-F3/17RB/17D/33. UNEXPLORED COMBINATORIAL HYPOTHESES IL family w.r.t STEAP3 IL-F3/17RB/17D/33 STEAP3 Table 12: 2nd order combinatorial hypotheses between STEAP3 and IL family members. 2.1.7. STEAP3-TFR family Ohgami et al. [2], show that the expression of STEAP2 and STEAP4 in erythropoietic tissues, their partial colocalization with TF and TFR1, and the demonstration that they155 have ferrireductase activity in vitro, indicate that STEAP2 and STEAP4 are reasonable candidates for redundant ferrireductases in the erythroid TF-cycle endosome. In colorectal cancer cells treated with ETC-1922159, some of the family members of TFR were down regulated, along with STEAP3. Individual recordings of these down regulations have been documented. I was able to rank 2nd order combination of TFR family160 members with STEAP3, that were down regulated. 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 TFR family w.r.t STEAP3. TFR2 - STEAP3 shows low ranking of 1063 (laplace) and 1169 (rbf). These rankings point to the synergy165 existing between the two components, which have been down regulated after the drug treatment. However, TFRC did not show any down regulation synergy with STEAP3. RANKING TFR FAMILY VS STEAP3 RANKING OF TFR FAMILY W.R.TSTEAP3 laplace linear rbf TFR2 - STEAP3 1063 2487 1169 TFRC - STEAP3 1946 663 2255 Table 13: 2nd order interaction ranking between STEAP3 VS TFR family members. One can also interpret the results of the table 13 graphically, with the following influences - •TFR family w.r.t STEAP3 with STEAP3 −>TFR-2. 9