Hyperglycosylated hcg drives malignancy in cancer cases.

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1 Review Article ttp:// Hyperglycosylated CG drives malignancy in cancer cases. Laurence A. Cole* USA CG Reference Service, 34 Broadmoor Way, P.O. Box 950 Angel Fire, New Mexico Abstract Purpose: Tropoblastic cancers (Coriocarcinoma, testicular and ovarian germ cell malignancies) secrete yperglycosylated CG and non-tropoblastic cancers secrete yperglycosylated CG free β- subunit.bot are structural variants of CG independent to te ormone CG. Tey are autocrines acting on a transforming growt factor β-ii (TGF-β-II), receptor not acting on te ormone receptor. Here tese molecules were examined as drivers of malignancy. Metods: Te cancer cell lines were examined, ow tey enzymatically invaded Matrigel cambers and ow teir growt responded to yperglycosylated CG and yperglycosylated CG free β-subunit signals. Use of yperglycosylated CG and yperglycosylated CG free β-subunit as tumor markers is examined in 959 cancer serum samples and 2257 cancer urine samples, plus 284 urines from benign disease patients. In addition, 56 new ovarian cancer urines were collected and tested in te supersensitive B204 assay. Results: Hyperglycosylated CG and its free β-subunit are bot strong promoters of malignancy functions. A total of 100 of tropoblastic malignancies produced yperglycosylated CG markers in serum and urine. A total of 30 of non-tropoblastic cancers were detected in serum and 44 in urine. As sown, in reality, considering simple and complex autocrine secretion, 100 of nontropoblastic malignancies produce yperglycosylated CG and yperglycosylated CG free β- subunit markers in urine. As demonstrated, yperglycosylated CG and its free β-subunit exist in all uman cancers and promote malignancy. Multiple oter researcers ave confirmed tese results. Discussion: It is inferred tat yperglycosylated CG and its free β-subunit are produced by all or most uman cancers, and are te promoters of malignancy. Keywords: Hyperglycosylated CG, Steroidogenesis, Ovulation, Luteogenesis. Accepted on 03 August, 2017 Introduction Wat promotes malignancy in uman cancers? A many times asked and never answered issue. Many autors ave suggested oncogenic proteins, suc as transforming growt factor β (TGF-β), TGF-β agonists and TGF-β antagonists. Te question still remains an issue and still remains unanswered [1-13]. Tis issue is addressed ere, cancer yperglycosylated CG and yperglycosylated CG free β- subunit, bot TGF-β antagonists, are seemingly te uman malignancy promoter tat differentiate benign and malignant cells. As proposed in tis article, cancer yperglycosylated CG is te most promising uman cancer malignancy promoter. Hyperglycosylated CG was discovered by me in 1997 [14]. It is a second form of CG, a TGF-β antagonist, yet a completely independent and separate molecule to te ormone CG. It sares an identical α-subunit and β-subunit amino acid sequence wit CG, and only differs from CG by aving tree or four type 2 O-linked oligosaccarides on te CG β-subunit C-terminal peptide, exasexaccarides and pentasaccaride verses tree of four sugar type 1, trisaccaride and tetrasaccaradide structures found on te ormone CG (Figure 1). Te tree dimensional structure of pregnancy and cancer yperglycosylated CG is virtually identical to te ormone CG (Butler SA, Computer+X-ray 3D Models (Figure 1) [15]. Hyperglycosylated CG is an autocrine binding a TGF-β-II receptor [16-18], and not acting at all on an CG/luteinizing ormone (LH) receptor like te ormone CG (Figure 1). Hyperglycosylated CG is made by invasive cytotropoblast cells during pregnancy, wile te ormone CG is made by fused syncytiotropoblast cells during pregnancy [19,20]. Hyperglycosylated CG acts on te autocrine cytotropoblast cell TGF-β-II receptor driving implantation of te blastocyst in te beginning of pregnancy [20,21], and deep implantation of emocorial placentation at 10 weeks gestation [22,23], and during te menstrual cycle finalizes ovulation [24]. In all tree processes, te autocrine TGF-β-II receptor drives invasive metalloproteinase and collagenase production [12,19], cell growt [19,25] and blocks cell apoptosis [26-28], tree malignancy-like steps. 1

2 Citation: Cole LA. Hyperglycosylated CG drives malignancy in cancer cases. J Mol Oncol Res. 2017;1(1):1-21. Figure 1. Te tree dimensional structure of te ormone CG (A), and te autocrines yperglycosylated CG (B), and cancer yperglycosylated CG (C). Butler computer and crystallograpy model Te yperglycosylated Hcg -TGF-β-II patway is te solitary enzymatic invasion patway and cellular growt patway coded in te uman genome. Cancer cells steal tis yperglycosylated CG and use it to drive uman malignancy, to drive enzymatic invasion, growt and blockage of apoptosis in cancer cells [26-28]. Tis is cancer yperglycosylated CG produced by tropoblastic cancers, and yperglycosylated CG free β- subunit produced by non-tropoblastic cancers [29], te molecules tat drives implantation in pregnancy, seemingly drives uman malignancy. Under medical dictionary definition, malignancy is te process of cancer advancement, cancer growt, and cancer tissue invasion. In te Merriam-Webster dictionary malignancy, it is defined as aggressively malicious, tending to produce deat or deterioration, tending to infiltrate, metastasize, and terminate fatally. As interpreted in tis article, malignancy means te process of cancer advancement, cancer growt promotion, and cancer tissue invasion. We test yperglycosylated CG and its free β- subunit for tese malignancy properties, and ow a malignancy promoter absolutely differentiates cancers and benign diseases. Te ability of tropoblastic cancers to produce yperglycosylated CG was confirmed and non-tropoblastic cancers produce yperglycosylated CG free β-subunit. Ten sow tat bot te molecules produced by cancers ave malignancy properties. Finally, te occurrence of tese tumor agents was examined to sow tat 100 of cancers produce tese malignancy agents. Materials and Metods Serum samples, 959, and urine samples 2257 were collected from men and women attending te oncology, toracic oncology and gynecologic oncology clinics at Yale New- Haven Hospital in New Haven CT. Samples collection was started in 1992, multiple tubes of serum and urine were libraried in -80 C Revco freezers wit te certification of Yale Clinical Researc Internal Review Board seeking full patient approval. Additionally, 283 urine samples from women and men wit benign disease were collected at Yale New Haven Hospital Oncology clinics and 154 serum and urine samples were at Oncology clinic from ealty and treated individuals. A furter group of 56 subjects wit ovarian cancer supplied urines in 1999 for super sensitive B204 test examination wit full IRB approval. At Yale University verbal consent was considered sufficient for collecting urine samples for tumor marker studies, signed consent was needed for serum samples. Cell lines, JEG-3 coriocarcinoma cell line, JAr coriocarcinoma cell line, NTERA testicular germ cell cancer, T24 epitelial bladder carcinoma cell line, KLE endometrial adenocarcinoma cell line, CaSki epidermoid cervical cancer cells, ScaBER squamous bladder cancer cell, Hec-1-a endometrial cancer cells, SK-MES-1 lung epitelial cancer cells and KM-H2 Hodgkin s lympoma cancer cells were all maintained continuously in culture in T75 flasks using ig glucose Dulbecco s modified Eagle s medium wit 10 fetal bovine serum. Cells were sub-cultured using 1X trypsin. In te experiment te cells were cultured in flasks 3-5 days in quadruplicate until tey reac approximately 70 flask confidence. Ten medium was canged and replaced wit medium containing 0 ng/ml, and yperglycosylated CG (JAr, JEG-3 and NTERA cell lines), or 0 ng/ml, and yperglycosylated CG free β-subunit (T24, KLE, CaSki, ScaBER, Hec-1-a, SK-MES-1 and KM-H2 cell lines). Cells were cultured for 24 and te cell number determined in eac flask using a emocytometer. Matrigel basement cambers were purcased from Corning Inc. (Corning, NY). Matrigel cambers were managed according to manufacturer s instructions, and manufacturer suggested calculation of proportion penetration. Cells, 70 confluent flasks of cells were suspended and transferred to Matrigel cambers, and cultured in Matrigel cambers for 24 and proportion penetration or invasion determined. Zero time culture cells were plated onto Matrigel s and control inserts (Biocoat Matrigel invasion s, BD Biosciences, Bedford, MA 01730), and cultured at 37ºC for 24 in Dulbecco s modified Eagle s medium wit 10 fetal bovine serum in quadruplicate. Matrigel s were processed and percentage invasion calculated. Briefly, s were reydrated in te incubator for 2 before use. Membranes and control inserts were ten plated (25,000 cells in 0.5 medium per plate). Table 1. Wat is produced by cancer cells? 2

3 Cole Cells JEG-3 coriocarcinoma cells Control cultures Control+100 ng/ml yperglycosylated CG NTERA testicular germ cells Control culture Control+100 ng/ml yperglycosylated CG CaSki epidermoid cervical cancer cells Control culture Control+100 ng/ml yperglycosylated CG β-subunit SK-MES-1 Epitelial lung cancer Control culture Control+100 ng/ml yperglycosylated CG β-subunit Effect (mean ± standard deviation) 48 ± 11 invasion of basement 88 ± 6.0 invasion of basement Ttest ± 12 invasion of basement 78 ± 8.1 invasion of basement Ttest ± 15 invasion of basement 69 ± 6.5 invasion of basement Ttest ± 11 invasion of basement 76 ± 6.9 invasion of basement Ttest 0055 Plates were cultured for 24, and s removed from inserts using a scalpel. Membranes were transferred to a slide using Cytoseal mounting medium (Stepens Scientific Inc., Riverdale NJ), exposing te under surface and invaded cells. Cells were stained wit DIF-Quick Stain (IMEB Inc., Cicago IL) to mark nuclei. Invaded cells were counted at 9 marked positions and were averaged for eac insert. Cell penetration or invasion of s is directly compared to tat of correspondingly cultured control inserts and percentage invasion calculated using te formula provided by te manufacturer. All samples were tested by immunometric 96 well microtiter plate assays using Immulon-1 flat bottomed plates. For B152 yperglycosylated CG and yperglycosylated CG free β- subunit plates were coated wit 1/1000 B152, or 15 µl antibody in 22 ml coating buffer. For B204 urine yperglycosylated CG free β-subunit plus β-core fragment plates were coated wit B204 4 mg/ml in 22 ml coating buffer. For te super sensitive B204 assay plates were coated at 0.40 mg/ml in 22 ml coating buffer. For te FBT11 free β-subunit assay plates were coated wit ml FBT11 antibody concentrate in 22 ml coating buffer. Te secondary antibody used was 4001-POD for all assays, a monoclonal antibody specific to core CG β-subunit. Coated plates were incubated wit 0.2 ml sample or standard and incubated 2 at room temperature wit saking for te B204 and FBT11 assay, 6 at room temperature for te super sensitive B204 assay, and 4 at room temperature for te B152 assay. In all 3 cases, te coating wit te peroxidase labeled secondary antibody, 4001 POD was 2 at room temperature. Finally, 200 µl of substrate (TMB reagent, dilute 50 wit water [Sigma part#t8665]) is added to eac well. After approximately 15 min incubation reaction is topped by te addition of 50 µl of 2 N HCl. Absorbance is ten read on te titerplate reader at 450 nm. Te B152 yperglycosylated CG and yperglycosylated CG free β-subunit assay is tested wit 5 C5 yperglycosylated CG standards at 2.0 fmol/ml, 8.0 fmol/ml, 32 fmol/ml, 128 fmol/ml and 512 fmol/ml. Te B204 assay is tested wit FEDE β-core fragment standard at 2.0 fmol/ml, 8.0 fmol/ml, 32 fmol/ml, 128 fmol/ml and 512 fmol/ml. Te super sensitive B204 assay uses standards at 0.1 fmol/ml, 0.4 fmol/ml, 1.6 fmol/ml, 6.4 fmol/ml and 25.6 fmol/ml. Te FBT11 assay is calibrated wit recombinant CG β-subunit standards at 4 ng/ml, 1 ng/ml, 0.24 ng/ml, 0.1 ng/ml and 0.04 ng/ml. Specificity studies sow tat te B152 assay detect yperglycosylated CG, yperglycosylated CG β-subunit, nicked yperglycosylated CG and nicked yperglycosylated CG β-subunit. Wit<0.2 detection of te ormone CG, and its dissociated CG β-subunit. Te B204 assay detects β-core fragment, CG free β-subunit, nicked CG free β-subunit, yperglycosylated CG β-subunit and nicked yperglycosylated CG β-subunit. Te FBT11 assay detects CG free β-subunit, nicked CG free β-subunit, yperglycosylated CG β-subunit and nicked yperglycosylated CG β-subunit [19]. Serum samples were also detected for total CG (CG, +nicked CG, +nicked CG missing β-subunit C-terminal peptide, +CG free β-subunit, +nicked β-subunit, +nicked β- subunit missing β-subunit C-terminal peptide, +yperglycosylated CG, +nicked yperglycosylated CG, +nicked yperglycosylated CG free β-subunit) using te automated Siemens Immulite assay. Tests were run according to manufacturer s instruction using a total CG standard mass curve, recombinant CG (4 ng/ml, 1 ng/ml, 0.25 ng/ml, 0.5 ng/ml, 0.2 ng/ml and 0.09 ng/ml), run wit every group of samples. All tumor marker data was entered into Microsoft Excel spreadseets and analyzed and sorted by diagnosis code. Results and Discussion 1. Wat do cancer cells secrete? Te freezers were searced for stored cancer patient serum and used for tumor marker studies and found 32 tropoblastic cancer (coriocarcinoma, ovarian germ cell and testicular germ cell cancers) serum samples and 92 non-tropoblastic cancer serum samples. All were tested for total CG in te Siemens Immulite assay, for yperglycosylated CG and yperglycosylated CG β-subunit in te B152 assay, and for 3

4 Citation: Cole LA. Hyperglycosylated CG drives malignancy in cancer cases. J Mol Oncol Res. 2017;1(1):1-21. free β-subunit verses α-β- dimer using te FBT11 assay (Table 1). Coriocarcinoma case 69, during terapy Table 2: Promotion of cancer cell invasion in quadruplicate 70 confluent Matrigel basement invasion cambers by C5 yperglycosylated CG and its β-subunit. Coriocarcinoma case 61, during terapy 1, pre-terapy Case or sample Immulite 1000 assay B152 assay B152 assay FBT11 assay 4, pre-terapy Standards Recombinant CG (Ovidrel), 1000 ng/ml Recombinant CG β-subunit, 600 ng/ml Hyperglycosylated CG standard C5, 1000 ng/ml C5 free β-subunit standard, 600 ng/ml Tropoblastic cancers Coriocarcinoma case 3, preterapy Coriocarcinoma case 7, preterapy Coriocarcinoma case 8, preterapy Coriocarcinoma case 11, preterapy Coriocarcinoma case 12, preterapy Coriocarcinoma case 23, preterapy Coriocarcinoma case 25, preterapy Coriocarcinoma case 26, preterapy Coriocarcinoma case 32, preterapy Coriocarcinoma case 36, during terapy Coriocarcinoma case 41, during terapy Coriocarcinoma case 42, during terapy Coriocarcinoma case 47, during terapy Coriocarcinoma case 48, during terapy , pre-terapy 6, pre-terapy 7, pre-terapy 11, pre-terapy case 7, pre-terapy case 9, pre-terapy case 10, pre-terapy case 11, pre-terapy case 14, pre-terapy case 17, pre-terapy Mean ± standard deviation Non-tropoblastic cancers ,364±43, ,427±4 3,729 Bladder cancer case ± ,492±4,227 Bladder cancer case Bladder cancer case Bladder cancer case Bladder cancer case Breast cancer case Breast cancer case Breast cancer case Breast cancer case Coriocarcinoma case 49, during terapy Breast cancer case Coriocarcinoma case 50, during terapy Breast cancer case Coriocarcinoma case 54, during terapy Coriocarcinoma case 57, during terapy Breast cancer case Breast cancer case

5 Cole Breast cancer case Cervical cancer case Cervical cancer case Cervical cancer case Cervical cancer case Cervical cancer case Cervical cancer case Cervical cancer case Cervical cancer case Cervical cancer case Cervical cancer case Endometrial cancer case Endometrial cancer case Endometrial cancer case Ovarian cancer case Ovarian cancer case Ovarian cancer case Ovarian cancer case Ovarian cancer case Vulvar cancer case Vulvae cancer case Mean 0.24 ± 0.29 Acetone concentrated pool of 58 cancer samples ± ± ± 0.31 Te standards are testsed first, recombinant CG (Serono) (Table 1). Tis is 100 ormone CG. As found, B152 assay only detected 0.13 confirming tat tis assay only detects yperglycosylated molecules, and does not detect te ormone CG. Secondly, te recombinant CG dissociated β-subunit was tested. As found, B152 only detected 0.11 confirming one again tat te ormone CG β-subunit is also not detected (Table 2). Ten te C5 yperglycosylated CG standard was tested, tis was 100 detected by te B152 assay, confirming tat tis assay is specific for yperglycosylated molecules. Finally, te assays were tested wit C5 yperglycosylated CG dissociated 0.1 β-subunit, tis was also 100 detected by te B152 assay. Similarly, it was found tat te FBT11 assay only detected a separated β-subunit, detecting recombinant CG dissociated β- subunit and C5 dissociated β-subunit. As publised previously [30], tropoblastic cancers produce cancer yperglycosylated CG, and non-tropoblastic cancers produce yperglycosylated CG free β-subunit. Cancer yperglycosylated CG and cancer yperglycosylated CG free β-subunit as a β-subunit wit triantennary N-linked oligosaccarides (Figure 1), wile pregnancy yperglycosylated CG as bi-antennary N-linked oligosaccarides. Te tropoblastic cancers produce a yperglycosylated CG was confirmed by Cole LA, and tat non-tropoblastic cancers produce a yperglycosylated CG free β-subunit. As sown (Table 1), pre-terapy, 32 tropoblastic cancers just produced yperglycosylated CG, B152 yperglycosylated CG assay mean detection 96 ± 12.5 of total CG. Non-tropoblastic cancers produced a free β-subunit (Table 1). 92 non-tropoblastic cancer serum samples were tested. Only 34 of te 92 cancer serum samples was detected by te Siemens Immulite total CG assay. Te balance were assumed to be producing low concentrations of tis tumor agent. Te B152 yperglycosylated CG and β-subunit assay detected 102 ± 6.2 of te total CG immunoreactivity in te remaining 34 serum samples. Tis indicated tat nontropoblastic cancers produce a yperglycosylated CG β- subunit. It is concluded tat all cancers secrete yperglycosylated CG or yperglycosylated CG β-subunit. As sown, bot yperglycosylated CG and yperglycosylated CG β-subunit bot act on a TGF-β-II receptor [16-18]. Tere was a concern about te 58 of 92 samples missed by tis testing as aving very low concentrations of yperglycosylated CG free β-subunit. Do tose producing low concentrations also produce primarily yperglycosylated CG free β-subunit. Te 58 samples were pooled, 3 ml of eac, tis gave me 174 ml. Using a metod described previously [14], te 174 ml was concentrated using 3X acetone. Tis precipitated te urine. Te precipitate was completely dissolved in 10 ml pospatebuffered saline buffer and ten tested as a 17.4X concentrate. Te concentrate was tested in te Immulite, B152 and FBT11 assays (Table 1). It contained 0.11 ng/ml according to te Siemens Immulite total CG assay, 0.12 ng/ml by te B152 yperglycosylated CG assay, and 0.10 ng/ml using te FBT11 free β-subunit assay. Tis confirmed tat te low concentration Samples also primarily contained a yperglycosylated CG free β-subunit. Many of te laboratories demonstrating tat non-tropoblastic cancers respond to yperglycosylated CG free β-subunit, or to te molecule secreted by te cancers [31-34], ave used CG β-subunit, not yperglycosylated CG β-subunit, to promote a cancer response. As publised, yperglycosylated CG is nicked and dissociated into a β-subunit to bind te TGF-β-II receptor [15]. Te receptor will respond to yperglycosylated CG β-subunit and to CG β-subunit [15]. Te LH-CG ormone receptor, owever will not respond to CG β-subunit. 5

6 Citation: Cole LA. Hyperglycosylated CG drives malignancy in cancer cases. J Mol Oncol Res. 2017;1(1):1-21. It was concluded tat tropoblastic cancers secrete cancer yperglycosylated CG dimer. It was also concluded tat all non-tropoblastic cancers primarily secrete a yperglycosylated CG free β-subunit. As demonstrated previously, bot cancer molecules act on a TGF-β-II receptor to elicit a growt, anti-apoptosis or invasion response [16,17,28]. 2. Cancer yperglycosylated CG and yperglycosylated CG free β-subunit Wy is yperglycosylated CG two molecules, yperglycosylated CG and yperglycosylated CG free β- subunit? Tropoblastic cancer produce yperglycosylated CG α-β subunit dimer, and non-tropoblastic malignancies produce yperglycosylated CG free β-subunit. Bot molecules are TGF-β-II receptor autocrines promoting cancer cell malignancy. Te reason needed to deal wit bot te dimer and te free β-subunit is very muc explained by Beebe et al. [29], and by Ruddon et al. [30]. To complete te syntesis of te β- subunit requires completion of 6 disulfide bridges. Te last two disulfide bridges, β and β93-100, requires a specialized disulfide isomerase, only found in tropoblast cells and pituitary cells. Witout tis isomerase, construction of te β- subunit is not completed, and β-subunit does not combine wit α-subunit. Tis produces a free β-subunit as made in non tropoblastic cancers. Only tropoblastic cancers make an α-β subunit dimer [29,30]. 3. Hyperglycosylated CG and yperglycosylated CG free β-subunit te malignancy molecules. Te malignancy properties of yperglycosylated CG and it free β-subunit were investigated by Cole LA. Multiple cancer cell lines were cultured on top of a Matrigel (Corning, Corning NY) basement and te extent of penetration of te by te cells determined. A wide variety of cancers was investigated (Table 3), ranging from coriocarcinoma cells to testicular germ cell malignancy cells, to cervical endometriod cancer cells, to lung epitelial cancer cells to Hodgkin lympoma cancer cells. In eac cell line control cells wit no additive placed in medium (cells produce yperglycosylated CG or its free β-subunit naturally) were compared to cells wit 100 ng/ml additive (Table 3). Table 3. Promotion of cancer cell 24 growt at 70 confluency by C5 yperglycosylated CG and its β-subunit. Cells Ttest vs 0 ng/ml Jar coriocarcinoma JEG-3 coriocarcinoma Supplement added to culture fluid effect on cell count Hyperglycosylated CG Hyperglycosylated CG Hyperglycosylated CG effect on cell count 112 cell 130 cell 110 cell P value NTERA Testicular germ cell Hec-1-a endometrial cancer ScaBER squamous bladder cancer KLE Endometrial adenocarcinoma SK-MES-1 Epitelial cancer KM-H2 lung T24 Bladder epitelial carcinoma Caski epitelial cervical carcinoma Hyperglycosylated CG Hyperglycosylated CG Hyperglycosylated CG Hyperglycosylated CG β-subunit Hyperglycosylated CG β-subunit Hyperglycosylated CG β-subunit Hyperglycosylated CG β-subunit Hyperglycosylated CG β-subunit Hyperglycosylated CG β-subunit Hyperglycosylated CG β-subunit Hyperglycosylated CG β-subunit Hyperglycosylated CG β-subunit Hyperglycosylated CG β -subunit Hyperglycosylated CG β -subunit Hyperglycosylated CG β -subunit Hyperglycosylated CG β -subunit Hyperglycosylated CG β -subunit 200 ng/m 128 cell 118 cell 132 cell 138 cell 166 cell count after cell 156 cell count after cell 132 cell 134 cell 163 cell 120 cell 145 cell 110 cell 128 cell 115 cell 142 cell P= Control cells penetrated te basement, 36 ± 12 to 49 ± 15. Additive cells penetrated te basement to a muc more significant extent 69 ± 6.5 to 88 ± 6.0. Eac cell line sowed a significant difference between control cultures and additive cultures. Multiple studies sow tat molecules acting on TGF-β-II receptors promote enzymatic cell to cell invasion by promoting production of metalloproteinases and collagenases [31-35]. It is concluded tat yperglycosylated CG and it free β-subunit 6

7 Cole drive cancer cell metalloproteinases and collagenases production. Te action of yperglycosylated CG and its free β-subunit on cancer grow te was investigated. Ten different cultured cancer cell lines were cultured to 70 confluency, and ten cultured wit yperglycosylated CG and yperglycosylated CG free β-subunit, and ten wit 200 mg/ml yperglycosylated CG and yperglycosylated CG free β- subunit supplement. Cell growt was compared to no supplement control cultures (Table 3). A wide variety of cells was investigated, ranging from Jar and JEG-3 coriocarcinoma cells, NTERA testicular germ cell cancer cells, Hec-1-a and KLE endometrial cancer cells, ScaBER and T24 bladder cancer cells, SK-MES-1 lung cancer, Caski cervical cancer and KM-H2 lympoma cells. Te addition of yperglycosylated CG and yperglycosylated CG free β- subunit caused 110 to 134 enancement in cell population, te addition of yperglycosylated CG and yperglycosylated CG free β-subunit caused a significantly greater (all additives caused a significant advancement in cell growt) enancement in cell population, 128 to 163 of te control population. Clearly, yperglycosylated CG and its free β-subunit enance cell growt and promote cell to cell invasion troug te actions of metalloproteinases and collagenases. Most of tese studies ave been repeated and confirmed by te 8 independent oter groups wo ave very muc confirmed our studies using a wide range of alternative cell lines [35-38]. Multiple oter autors ave also sown tat yperglycosylated CG and it free β-subunit also blocks apoptosis, anoter action in malignancy [39]. All told, yperglycosylated CG and yperglycosylated CG free β-subunit promote te production of te invasive enzymes collagenases and metalloproteinases, promote cell growt, and block cellular apoptosis, or ave all malignancy properties. Having sown tat yperglycosylated CG and yperglycosylated CG β-subunit promote all malignancy properties, it was necessary to sow te occurrence of tese tumor agents. 4. Tumor markers Te occurrence of yperglycosylated CG and its free β- subunit as cancer markers were investigated (Table 4). Te serum yperglycosylated CG and its free β-subunit were measured using te B152 immunoassay, and te urine degradation products of yperglycosylated CG and yperglycosylated CG β-subunit, te terminal degradation product β-core fragment using te B204 immunoassay. Te B204 immunoassay detects β-core fragment and nicked yperglycosylated CG β-subunit. Te serum and urine on 110 tropoblastic cancer cases were examined, and serum on 849 non-tropoblastic cancer cases, and urine on 2,167 nontropoblastic cancer cases (Table 4). Table 4. Serum yperglycosylated CG+β-subunit (B152 assay) and urine degradation product yperglycosylated CG β-subunit+β-core fragment (B204 assay) as tumor markers. Source Tropoblastic malignancies Serum, B152 assay #Cas es Coriocarcinoma Ovarian germ cell cancer #Detec ted Sensitiv ity Urine assay #Cas es B204 #Positi ve Sensitiv ity Total Non-tropoblastic malignancy Bladder cancer Breast cancer Cervical cancer Colorectal cancer Endometrial cancer Gastric cancer Hepatic cancer Lung cancer Intestinal cancer Lympoma Ovarian cancer Pancreatic cancer Prostate cancer Renal cancer Uterine cancer Vulvar cancer Total Healty NED, post cancer cemoterapy NED, post cancer surgery Healty female, no cancer istory Healty male, no cancer istory Total Benign Disease Benign gynecological lesion, tumor Benign lung lesion Follicular ovarian cyst, benign

8 Citation: Cole LA. Hyperglycosylated CG drives malignancy in cancer cases. J Mol Oncol Res. 2017;1(1):1-21. Benign endometrial wart Ovarian Endometrioid carcinoma IV Persistent 0.12 Benign ovarian cyst, nonfunctional Ovarian Endometrioid carcinoma IV treated 0.15 Benign prostate yperplasia treated 0.2 Cervical carcinoma in-situ Cervical dyskaryosis Condyloma Endometriosis Myoma Total As found, 100 of tropoblastic cancer case were detected in serum and urine (Table 4). In contrast, an average of 30 of non-tropoblastic cancer cases were detected in serum, and 44 of cases in urine samples. Te iger detection in urine may be due to te rapid blood clearance of yperglycosylated CG free β-subunit and its degradation products. Detection of non-tropoblastic cancers varied greatly according to cancer site. In serum, detection ranged from 17 wit colorectal cancers to 38 wit ovarian cancers. In urine, using te B204 assay, detection ranged from 34 wit breast cancers to 70 wit ovarian malignancies (Table 4). Questions ave been raised on wy 34 to 70 and not 100, since 100 of cancers sow malignancy? 5. Simple and complex autocrines Tere are two kinds of TGF-β autocrines, simple autocrines and complex autocrines [40-43]. Simple autocrines are produced at low concentration, are secreted and act directly on te cancer cell TGF-β-II receptor witout necessarily circulating [44] Complex autocrines are produced at iger concentration. Tey are secreted, and circulate in te blood stream before acting back on te original cancer cell TGF-β-II receptor. Are te cancers simple autocrine or complex autocrine TGF-β-II receptor cases? Does simple autocrine production explain te lower detection, in nontropoblastic cancers (Table 4)? To address tis issue 56 new non-treated ovarian cancer cases were tested providing urine samples wit te B204 assay. Te current assay used as a sensitivity of 1.4 fmol/ml, and te cut off in te tumor marker assay of 3.0 fmol/ml. A significantly more sensitive assay was developed by 10-fold reducing te plating concentration of B204 antibody, and by extending te first incubation time from 2 to 6. Te new super-sensitive assay detected down to 0.08 fmol/ml, and as a tumor marker cut-off of 0.10 fmol/ml. Tis assay was used to test te 56 ovarian cancer urines Table 5. Table 5. Ovarian cancer super-sensitive urine B204 assay. Tumor marker study of 56 ovarian cancer cases receiving no terapy using super-sensitive B204 assay (sensitivity>0.1 fmol/ml). Persistent 0.2 Recurrent 0.25 IV Recurrent 0.4 IV Recurrent 0.4 Ovarian Brenner tumor Ia treated 0.58 treated 0.6 New 0.75 Ovarian Brenner tumor Ia treated 0.88 Ovarian Serous cystadenocarcinomacucystcancerccancercysta denocarcinoma Recurrent 1.2 Serous cystadenocarcinoma II Recurrent 1.6 Recurrent 2.1 Ovarian Endometrioid carcinoma III Recurrent 2,3 Persistent 2.5 Ovarian Mucinous ccarcinomacarcinomacystadenocarcinoma Ia Ovarian Endometrioid carcinoma I treated 2.9 treated 3.1 Ovarian Granulosa-teca cell malignancy Recurrent 3.1 II treated 3.5 Ovarian Granulosa-teca cell malignancy Persisent 3.7 Ovarian Mucinous cystadenocarcinoma III Recurrent 4.2 III New 4.5 Ovarian Clear cell carcinoma III treated 4.3 treated 4.8 Ovarian Clear cell carcinoma Recurrent 5.5 New 6.3 Ovarian Mixed epitelial tumor treated 6.6 treated 7.1 Ovarian Mixed epitelial tumor III New 7.9 treated 8.1 Persistent 8.8 Patologic Diagnosis Stag e Status β-core fragment treated 9 8

9 Cole III Persistent 9.5 Ovarian Mixed epitelial tumor Ovarian Mixed epitelial tumour IIc IIb treated 10 treated 11.3 III New 11.4 IV Recurrent 11.4 IV Recurrent 12 Recurrent 12 Recurrent 12 Ovarian Endometrioid carcinoma IIc treated 12.2 Ovarian Endometrioid carcinoma III Persistent 13.1 Ovarian Mixed mesodermal carcinoma III Recurrent 14.4 Ovarian Mixed mesodermal carcinoma III Recurrent 16.3 IV Recurrent 16.9 IV Recurrent 17.5 treated 18.8 Persistent 20 Ovarian Mixed epitelial tumor Recurrent 20 Ovarian Mixed epitelial tumor III Recurrent 21 IV IV treated 28 treated 29 III New 32 IIb treated 41 Ovarian Malignant dermoid cyst/teratoma IIb New 54 As sown in Table 5, 56 of 56 cancer urines were positive using te super-sensitive B204 assay. Tirty-nine (70) of te urines would be detected by a tumor marker assay wit 3.0 fmol/ml cut-off, and 56 of 56 urine (100) would be detected in te assay using te new 0.1 fmol/ml cut-off. It was concluded tat tirty-nine cases probably produced a complex autocrine (>3.0 fmol/ml cut-off samples), and te balance, seventeen cases, probably produced tiny concentrations (<3.0 fmol/ml), or were simple autocrines. It was clear tat 100 of cases produced yperglycosylated CG and its free β-subunit. Seemingly 100 of all cancers, as simple autocrine and complex autocrine TGF-β-II cancers. In support of te concept tat all cancers produced simple and complex autocrines, Acevedo et al. [45-47] used flow cytometry to sensitively examine -associated CGrelated molecules produced by cancer cells. Eac sowed and confirmed, as indicated in Table 5 tat 100 of cancer cells produced te tumor markers. It was concluded tat in reality 100 of all cancer produce yperglycosylated CG and its free β-subunit. 6. Benign diseases If yperglycosylated CG and its free β-subunit control cancer cell malignancy, ten tese molecules sould not be expressed in benign diseases. 284 urines from women wit a wide variety of benign diseases were tested, using te B fmol/ml cutoff assay (Table 4). None of te 284 benign disease urines contained B204 reactive materials. Conclusions It was concluded tat 100 of cancers produce yperglycosylated CG or its free β-subunit, and tat no benign disease cases are positive for tis malignancy promoter. It was also concluded tat expression of te CG β-subunit gene by cells marked cancer, and syntesis of yperglycosylated CG or yperglycosylated CG free β- subunit made a cell malignant, or a cancer cell. References 1. Miller DM, Blume S, Borst M, et al. Oncogenes, malignant transformation, and modern medicine. Am J Med Sci. 1990;300: Croce CM. Oncogenes and cancer. N Engl J Med. 2008;358: Yokota J. Tumor progression and metastasis. Carcinogenesis. 2000;21: Martin S. Te Hunting of te Src, G. Nature Rev Molec Cell Biol. 2001;2: Todd R, Wong DT. Oncogenes. Anticancer Res. 1999;19: Summy JMM, Gallick GE. Src family kinases in tumor progression and metastasis. Cancer and Metast Rev. 2003;22: Bos JL. ras oncogenes in uman cancer: a review. Cancer Researc. 1989;49: Reiss M. TGF-beta and cancer. Microbes Infect. 1999;1: Bierie B. TGF-β and cancer. Cytokine Growt Factor Rev. 2006;17: decaestecker MP, Piek E, Roberts AB, et al. J Natl Cancer Inst. 2000;92: Akurst RJ, Balmain A. Genetic events and te role of TGF beta in epitelial tumour progression. J Patol. 1999;187: Murpy G, Reynolds JJ, Witam SE, et al. Transforming growt factor beta modulates te expression of collagenase and metalloproteinase inibitor. Euro MoleSc Biol Org J. 1987;6: Akurst RJ. TGF-β antagonists: Wy suppress a tumor suppressor? J Clin Invest. 2002; 109: Elliott MM, Kardana A, Lustbader JW, et al. Carboydrate and Peptide structure of te α- and β-subunits of uman corionic gonadotropin from normal and aberrant 9

10 Citation: Cole LA. Hyperglycosylated CG drives malignancy in cancer cases. J Mol Oncol Res. 2017;1(1):1-21. pregnancy and coriocarcinoma. EndocrinE. 1997;7: Cole LA. Te difference between te ormone CG and te autocrine yperglycosylated CG. Biosci Reports Butler SA, Ikram MS, Matieu S, et al. Te increase in bladder carcinoma cell population induced by te free beta subunit of CG is a result of an anti-apoptosis effect and not cell proliferation. Brit J Cancer. 2000;82: Berndt S, Blacer S, Munuat C, et al, Hyperglycosylated uman corionic gonadotropin stimulates angiogenesis troug TGF-β receptor activation. FASEB J. 2013;12: Amud F, Gos S, Sina S, et al. TGF-β-induced CG-β regulates redox omeostasis in glioma cells. Molec Cellul Biocem. 2015;399: Cole LA, Dai D, Leslie KK, et al. Gestational tropoblastic diseases 1 Patopysiology of yperglycosylated CG-regulated neoplasia. Gynecologic Oncology. 2006;102: Sasaki Y, Ladner DG, Cole LA. Hyperglycosylated CG te source of pregnancy failures. Fertil Steril. 2008;89: Evans J, Salamonsen LA, Menkorst E, et al. Dynamic canges in yperglycosylated uman corionic gonadotropin trougout te first trimester of pregnancy and its role in early placentation. Hum Reprod. 2015;30: Cole LA, Brennan MC, Hsu C-D, et al. Hyperglycosylated CG, A Sensitive Screening Test For Preeclampsia / Gestational Hypertension. Preg Hypertension Baado-Sing RO, Oz AU, Kingston JM, et al. Te role of yperglycosylated CG in tropoblast invasion and te prediction of subsequent pre-eclampsia. Prenat Diagn. 2002;22: Cole LA. Ovarian yperglycosylated CG and pituitary sulfated CG bot climax wit te menstrual mid-cycle LH peak. Matern Cild Healt J Iles RK. Ectopic CGβ expression by epitelial cancer: Malignant beavior metastasis and inibition of tumor cell apoptosis. Molec Cellul Endocrinol. 2007; Jankowska A, Gunderson SI, Warcol JB, et al. Reduction of CGβ subunit expression by modified U1 snrna caused apoptosis in cervical cancer cells. Molec Cancer. 2008;7: Li D, Wen X, Gali L, et al. CG β expression by cervical squamous carcinoma-in vivo istological association wit tumour invasion and apoptosis. Histopatol. 2008;53: Cole LA, Butler SA. Hyperglycosylated CG, CGβ and Hyperglycosylated CGβ Intercangeable Cancer Promoters. Molec Cellul Endcrinol. 2012;349: Cole LA. CG and Hyperglycosylated CG Carboydrate Structures Corrected. J Glycobiol. 2014;3: Beebe JS, Hut JR, Ruddon RW. Combination of te corionic gonadotropin free beta-subunit wit alpa. Endocrinol. 1990;126: Ruddon RW, Serman SA, Bedows E. Protein folding in te endoplasmic reticulum: Lessons learned from te uman corionic gonadotropin β-subunit. Protein Science 1996;5: Murpy G, Reynolds JJ, Witam SE, et al. Transforming growt factor beta modulates te expression of collagenase and metalloproteinase inibitor. Euro Molec Biol Org J. 1987;6: Urı a JA, Jiménez MG, Balbı n M, et al. Differential Effects of Transforming Growt Factor-β on te Expression of Collagenase-1 and Collagenase-3 in Human Fibroblasts. J Biol Cem. 1998;273: Yamane K, In H, Asano Y, et al. Antagonistic effects of TNF-alpa on TGF-beta signaling troug downregulation of TGF-beta receptor type II in uman dermal fibroblasts. J Immunol. 2003;171: Quresi Y, Sylvester J, Mabrouk E, et al. TGF-?-induced expression of tissue inibitor of metalloproteinases-3 gene in condrocytes is mediated by extracellular signalregulated kinase patway and Sp1 transcription factor. J Cellular Pysiol. 2005;203: Jankowska A, Gunderson SI, Warcol, JB. Reduction of CGβ subunit expression by modified U1 snrna caused apoptosis in cervical cancer cells. Molec Cancer. 2008;7: Gillott DJ, Iles RK, Card T. Te effects of beta-uman corionic gonadotropin on te in vitro growt of bladder cancer cell lines. Br J Cancer. 1996;73: Guo X, Liu G, Scauer IG, et al. Overexpression of te β Subunit of Human Corionic Gonadotropin Promotes te Transformation of Human Ovarian Epitelial Cells and Ovarian Tumorigenesis. Am J Patol. 2011;179: Hamade AL, Nakabayasi K, Sato A, et al. Transfection of antisense corionic gonadotropin β gene into coriocarcinoma cells suppresses te cell proliferation and induces apoptosis. J Clin Endocrinol Metab. 2005;90: Isklander YL, Byrant JL, Zemn RA, et al. Tumorigenesis and metastasis of neoplastic Karposis s Sarcoma cell line in immunodeficient mice blocked by a uman pregnancy ormone. Nature. 1995;375: Cole LA, Butler SA. B152 anti-yperglycosylated uman corionic gonadotropin free β-subunit. A new, possible treatment for cancer. J Reprod Med. 2015;60: Offermann MK, Faller DV. Autocrine induction of major istocompatibility complex class I antigen expression results from induction of beta interferon in oncogene transformed BALB/c-3T3 cells. Mol Cell Biol. 1989;9: Ikusima H, Miyazono K. TGFβ signalling: a complex web in cancer progression. Nature Reviews Cancer. 2010;10: Gold LI, Saxena B, Mittal KR, et al. Increased Expression of Transforming Growt Factor β Isoforms and Basic Fibroblast Growt Factor in Complex Hyperplasia and Adenocarcinoma of te Endometrium: Evidence for 10

11 Cole Paracrine and Autocrine Action. Cancer Res. 1994;54: Zeinoun Z, Teugels E, Vermeij J, et al. Restoration of an impaired TGF-beta1 autocrine growt-inibitory circuit results in growt inibition of ovarian epitelial cancer cells and complete inibition of teir tumorigenicity. Cancer Epidemiol. 2003;27: Munir S, Xu G, Wu Y, Yang B et al. Nodal and ALK7 inibit proliferation and induce apoptosis in uman tropoblast cells. J Biol Cem. 2004;279: Acevedo HF, Kricevsky A, Campbell-Acevedo EA, et al. Flow cytometry metod for te analysis of associated uman corionic gonadotropin, its subunits, and fragments on uman cancer cells. Cancer. 1992;69: *Correspondence to Laurence A. Cole USA CG Reference Service, 34 Broadmoor Way, P.O. Box 950Angel Fire, New Mexico Tel: larry@cglab.com 11

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