Production of Pregnancy-Associated Plasma Protein-A (PAPP-A) by Cultured Tumour Granulosa Cells

The cGAS-STING pathway has established itself as a critical innate immune pathway that has the ability to significantly affect tumor initiation and progression. The expression, methylation, immunological functions, and prognostic importance of cGAS-STING pathway-related genes in cervical squamous cancer (CESC) patients have not yet been thoroughly elucidated. First, we explored the expression of cGAS and STING in cervical carcinoma samples from TCGA by comparing the mRNA and protein levels of cGAS and STING in both TCGA cervical tumor patient samples and cervical tumor cell lines. Second, we examined the CD4+T and CD8+T cell infiltration in STING high and low samples and made Kaplan-Meier prognosis analysis of STING protein expression. Third, to verify the findings in TCGA public datasets, we retrospectively selected 40 cervical squamous carcinoma patients and 10 normal cervical tissues and evaluated cGAS and STING protein expression using immunohistochemistry (IHC). All patients have detailed clinical information, which includes age, FIGO stage, menstruation status, follow-up time, histology, tumor diameter, and serum tumor markers. In both cervical tumor patient samples and cell lines, we observed that cGAS is increased, whereas STING is decreased in tumors, which leads to decreased CD4+T and CD8+T cell infiltration and poor prognosis. Furthermore, the cGAS mRNA transcript showed a gradual increase and STING mRNA showed a decrease according to the tumor stage, tumor grade, metastasis status, and histology types. We confirmed the expression of cGAS and STING proteins in clinical cervical tumor samples using IHC. Mechanically, cGAS and STING showed different DNA methylation patterns, which might contribute to the differences in cGAS and STING mRNA and protein levels. Our work identified different expressions and methylation patterns of cGAS and STING in cervical cancer and their correlation with immune T cell infiltration and prognosis. More mechanistic study is needed to understand the cGAS-STING pathway in cervical squamous tumor.

Overexpression of epidermal growth factor type-1 receptor (EGF-R1) in cervical cancer: Implications for Cetuximab-mediated therapy in recurrent/metastatic disease

To discover the value of combined maternal first and second-trimester serum β-hCG, pregnancy associated plasma protein A (PAPP-A), alpha-fetoprotein(AFP)and unconjugated estriol (uE3) in the prediction of preeclampsia. A total of 1 805 pregnant women who had antenatal care at International Peace Maternal and Child Health Hospital Affiliated to Shanghai Jiaotong University between April 2012 and June 2013 were selected prospectively by random method. According to the outcome, they were defined as the control group and the preeclampsia group (including mild and severe cases). PAPP-A and β-hCG level were measured at 10-14 gestational weeks. AFP, β-hCG and uE3 were measured at 15-20 gestational weeks. The relevance between the serological indicators and outcomes was analyzed. The value of the indicators was judged by receiver operating characteristic (ROC) and Youden index, and the relevant predictive boundary values were identified. (1) Among the 1 805 cases, 1 739 women did not have hypertension(the control group), while 66 women had preeclampsia (the preeclampsia group). The incidence of preeclampsia was 3.66% (66/1 805), including 43 mild cases and 23 severe cases. (2) At 10-14 gestational weeks, the mean value of PAPP-A in the control group was (3 972 ± 2 311) mU/L, while in the preeclampsia group it was (2 837 ± 1 849)mU/L. The difference between the two groups had statistical significance (P < 0.01). The mean value of β-hCG of the control group was 55(37∼83) U/L, while in the preeclampsia group it was (57 ± 35)U/L. There was no statistical significance (P > 0.05). PAPP-A, β-hCG and AFP of mild preeclampsia cases were (3 249 ± 1 877) mU/L, (61 ± 38) U/L and (35 ± 11) µg/L respectively, and in severe cases they were(1 758 ± 1 297)mU/L, (47 ± 23)U/L and (47 ± 22)µg/L, respectively. There was statistically significant difference in PAPP-A (P < 0.05). (3) At 15-20 gestational weeks, β-hCG, AFP and uE3 in the preeclampsia group were (47 909 ± 31 396 )U/L, (38 ± 15)µg/L and (0.98 ± 0.31)µg/L respectively, and in the control group they were (39 267 ± 25 054 )U/L, (47 ± 18)µg/L and (1.17 ± 0.39) µg/L, respectively. AFP and uE3 of the preeclampsia group were lower than those in the control group and the difference was statistically significant (P < 0.05). However, β-hCG and uE3 of the mild preeclampsia cases and the severe cases had no statistical difference (P > 0.05). (4)At 10-14 gestational weeks, PAPP-A demonstrated positive relevance to the newborn weight (r = 0.068, P = 0.011) and gestational weeks at delivery (r = 0.057, P = 0.048). At 15-20 weeks, positive relevance was found between AFP and the newborn weight (r = 0.149, P = 0.000), while negative relevance was found between β-hCG and Apgar scores (r = -0.085, P = 0.024), and positive relevance was found between uE3 and gestational weeks at delivery (r = 0.086, P = 0.036). (5) PAPP-A, AFP and uE3 data were used as testing parameters to obtain the boundary values of preeclampsia prediction as follows: PAPP-A 1 831 mU/L, AFP 41 µg/L and uE3 1.04 µg/L. The specificity was 97.82% , 98.54% and 98.80% , respectively. (6) ROC was drawn and Youden index was calculated based on the joint predicative factor of PAPP-A, AFP and uE3. Youden index reached its peak (0.41) when the joint predictive factor was 0.032, meaning that the factor had the highest prediction value. The prediction value of the PAPP-A, AFP and uE3 was 0.032, with the specificity and sensitivity of 98.93% and 70.59%, respectively. The odds ratio was 2.37. Both the individual parameter (PAPP-A, AFP and uE3) and the combined data have prediction value for preeclampsia, but the latter is more effective than any of the single parameter.

Immunohistochemical analysis of proliferation and differentiation in organotypic cultures of cervical tumor cell lines

We have recently reported that there is a significant Raf-1 kinase dependency of paclitaxel resistance in human cervical tumor cell lines. In light of the possibility that Raf-1 kinase inhibitors could be used to enhance paclitaxel responsiveness in ovarian cancer, we have characterized the Raf-1 kinase dependency of paclitaxel resistance in ovarian cancer cells. The relationship between Raf-1 kinase activity and the sensitivity to clinically relevant paclitaxel concentrations was determined in four ovarian cancer cell lines (CA-OV3, SK-OV3, 2780/WT and OAW42/WT). Furthermore, in recognition that such a drug combination would initially be used in patients whose tumors have recurred following cisplatin/paclitaxel treatment, we also determined the Raf-1 kinase dependency of paclitaxel cytotoxicity in cisplatin resistant variants of two of the ovarian cell lines (2780/CP and OAW42/CP). In the two cell lines (2780/WT and OAW42/WT) that possess a wild-type TP53 (TP53wt), the relationship between Raf-1 kinase activity and paclitaxel resistance was different from that observed in the cervical tumor cell lines. In these cell lines, paclitaxel-induced far more cell killing than would have been predicted from their Raf-1 kinase activity. However, in the ovarian cancer cell lines (CA-OV3, SK-OV3, 2780/CP and OAW42/CP) that have a mutant TP53 (TP53mut), the cytotoxicity induced by 60 nM paclitaxel exhibited the same relationship to Raf-1 kinase activity as previously observed in cervical tumor cell lines. These data suggest that the therapeutic efficacy of paclitaxel in ovarian cancer patient whose tumors have TP53mut might be increased if it is administered in combination with Raf-1 kinase inhibitors, e.g., ISIS 5132.

Docetaxel (Taxotere) is becoming increasingly important in the treatment of many tumor sites and is unusually active in tumors that are resistant to the structurally similar taxane, paclitaxel. These data suggest that the processes that confer cellular paclitaxel resistance may have a substantially lower impact upon the cytotoxicity induced by docetaxel. We have recently reported that there is a marked Raf-1 kinase dependency of paclitaxel resistance in human cervical and ovarian tumor cell lines. We therefore characterized the impact that inherent and genetically induced variations in Raf-1 kinase activity have on the docetaxel cytotoxicity in human ovarian and cervical cancer cell lines. Our data suggest that docetaxel cytotoxicity is independent of Raf-1 kinase activity in the cell lines studied and that the lack of cross-resistance between these two taxane compounds may be due to the differential impact that Raf-1 kinase activity has on their cytotoxicity. Should these relationships pertain to the clinical situation, these findings could form the basis for a molecular-based triage of patients to receive docetaxel when response to paclitaxel may be unlikely due to high Raf-1 kinase activity.

We have recently demonstrated that overexpression of dihydrodiol dehydrogenase (DDH) in human ovarian carcinoma cells (2008/C13*) is associated with cisplatin and carboplatin resistance. Furthermore, we have also elucidated that transfection of parental human ovarian carcinoma cells with a full-length DDH1 cDNA leads to induction of resistance to the platinum drugs. The development of cisplatin resistance in the transfected cells is associated with an increase in DDH enzyme activity. Previous studies have identified several different mechanisms for development of cisplatin resistance, including altered DNA repair capacity, increased GSH-based detoxification, and increased metallothionein content. However, none of these mechanisms has been found to be universally associated with the development of cisplatin resistance in tumor cells from different tissue sources. The present study was undertaken to assess whether overexpression of DDH1 or DDH2 (in human ovarian, cervical, lung and germ-cell tumor cell lines) could specifically induce resistance to the platinum drugs in these cell lines. We demonstrated a ubiquitous association of increased expression of DDH1 or DDH2 (as judged by increased enzyme activity in transfected clones) with development of resistance to cisplatin and carboplatin. Moreover, we also found a lack of cross-resistance to anticancer drugs that have a different mode of action including paclitaxel, vincristine, doxorubicin hydrochloride, and melphalan. Although at present it is not clear how DDH is involved in platinum drug resistance, the identification of this gene as a causal factor in a series of cell lines derived from different tumors with different intracellular compositions indicates the importance of deciphering this hitherto undefined pathway which can produce resistance to platinum drugs.

Metformin is one of the most widely prescribed antidiabetics for type 2 diabetes. A critical role of metformin against tumorigenesis has recently been implicated, although several studies also reported the lack of anticancer property of the antidiabetics. Given the controversies regarding the potential role of metformin against tumour progression, the effect of metformin against breast, cervical, and ovarian tumour cell lines was examined followed by in vivo assessment of metformin on tumour growth using xenograft breast cancer models. Significant inhibitory impact of metformin was observed in MCF-7, HeLa, and SKOV-3 cells, suggesting an antiproliferative property of metformin against breast, cervical, and ovarian tumour cells, respectively, with the breast tumour cells, MCF-7, being the most responsive. In vivo assessment was subsequently carried out, where mice with breast tumours were treated with metformin (20 mg/kg body weight) or sterile PBS solution for 15 consecutive days. No inhibition of breast tumour progression was detected. However, tumour necrosis was significantly increased in the metformin-treated group, accompanied by decreased capillary formation within the tumours. Thus, despite the lack of short-term benefit of metformin against tumour progression, a preventive role of metformin against breast cancer was implicated, which is at partially attributable to the attenuation of tumour angiogenesis.

We have previously demonstrated that overexpression of dihydrodiol dehydrogenase isoform 1 (DDH1) or DDH2 leads to the induction of drug resistance to platinum based drugs in human ovarian, lung, cervical and germ cell tumor cell lines. DDH belongs to a family of aldoketo reductases that are involved in the detoxification of several endogenous and exogenous substrates. DDH1 and DDH2 in particular have been shown to be involved in the detoxification (activation?) of polycyclic aromatic hydrocarbons (PAH). Based on the involvement of DDH in the detoxification of electrophilic PAH intermediates, the effect of DDH on the production of reactive oxygen species (ROS) in a cisplatin-sensitive and -resistant human ovarian carcinoma cell line was investigated in the current study. In addition to the overexpression of DDH1 and DDH2, increased expression of DDH3 was demonstrated in the cisplatin-resistant 2008/C13* cells, compared to the parental 2008 cells. However, as assessed by RT-PCR, neither cell line expressed DDH4. The 2008/C13* cells were eightfold resistant to cisplatin, and transfection experiments utilizing cisplatin-sensitive 2008 cells suggest that this could be mediated by overexpression of either DDH1, DDH2, or DDH3. The 2008/C13* cells had lower basal intracellular ROS level as compared to the 2008 cells and ROS production was decreased in the recombinant 2008 cells with forced, constitutive overexpression of either, DDH1, DDH2, or DDH3. Transfection of siRNA against DDH1 or DDH2 in the cisplatin-resistant 2008/C13* cells not only significantly decreased their cisplatin-resistance index (as assayed by MTT and colony formation assay) but also led to an increase in the basal levels of ROS production (although transfection of siRNA against DDH3 resulted in cell death). The 2008/C13* cells were found to be cross-resistant to the cytotoxic effects of hydrogen peroxide and tert-butyl hydroperoxide and knockdown of either DDH1 or DDH2 expression (using siRNA) resulted in sensitization of the resistant cells to these agents. These results support the conclusion that the increased levels of DDH in the 2008/C13* cells are directly responsible for the reduced production of ROS and that this may play a role in the development of cisplatin resistance.

TNF-like weak inducer of apoptosis (TWEAK) and FGF-inducible 14 (Fn14) are a TNF superfamily ligand-receptor pair involved in inflammation, oncogenesis, tumor invasion, migration, survival and resistance to chemotherapy. The Fn14 receptor is expressed at relatively low levels in normal tissues, but is known to be dramatically elevated in a number of tumor types, including brain and breast tumors. Thus, the TWEAK/Fn14 axis appears to be an excellent candidate for therapeutic intervention. We have developed an immunoconjugate designated ITEM4-rGel containing a high-affinity anti-Fn14 monoclonal antibody conjugated to recombinant gelonin (rGel), a highly cytotoxic, ribosome-inactivating n-glycosidase. The ITEM4-rGel conjugate was generated and purified and contained no contaminating free antibody or rGel. The final material contained both antibody + 1 rGel (major) and antibody + 2 rGel (minor) species. We analyzed Fn14 expression in human tumor cell lines using flow cytometry and Western blot analysis. Fn14 was expressed in a variety of tumor lines including breast, brain, bladder, skin, lung, ovarian, pancreatic, colon, prostate, and cervical tumor cell lines. Both ITEM4 and ITEM4-rGel were found to bind to cells to an equivalent extent. Confocal immunofluoresence studies showed that ITEM4-rGel specifically and rapidly (within 2 hrs) internalized into MDA-MB-231 breast cancer cells and T-24 bladder cancer cells but not into Fn14-deficient mouse embryonic fibroblasts. Cytotoxicity studies against 22 different tumor cell lines showed that ITEM4-rGel was highly cytotoxic to Fn14-expressing cells (IC50 ranged from 0.8 pM-25 nM) and was 50 to 0.5 ×106 fold more potent than free rGel. Minimum contact time studies showed that as little as 12 hr exposure achieved maximal cytotoxic effect. Mechanistic studies showed that ITEM4-rGel induced HMGB1 release following treatment of MDA-MB-231, T-24, AAB 527, and BxPC-3 cells. In addition, target cells showed induction of apoptosis, as measured by Annexin V staining and caspase-3 cleavage. ITEM4-rGel treatment also induced the non-canonical NF-κB pathway, up-regulated the tumor suppressor protein p53 and down-regulated survivin expression. Preliminary mouse xenograft tumor model studies are ongoing. These data suggest that the ITEM4-rGel construct may warrant further development as a novel therapeutic agent against a broad range of solid tumor types. Research conducted, in part, by the Clayton Foundation for Research (MGR), and supported by NIH grant NS55126 (JAW) and DOD Breast Cancer Concept Award BC086135 (JAW). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2574.

Emerging studies have clarified the critical role of LncRNA MALAT1 in various pathological progressions. Here, we identified its positive relationship with cervical carcinoma proliferation. Cervical carcinoma has been considered as one of the most malignant tumors among female. Thus, our study was designed to investigate the underlying mechanism of LncRNA MALAT1 on cervical tumor cell proliferation. We observed that miR-124 was the potential target of LncRNA MALAT1 in cervical tumor cell lines (Hela, C-33A, Caski, and SiHa), the expression level of which is negatively correlated with LncRNA MALAT1 in cervical tumor cells, tissues of cervical patients, and mice. Gain- or loss-of-function analyses in cervical tumor cells have further verified the regulatory role of MALAT1 on miR-124. Additionally, the proliferation of cervical carcinoma was inhibited by miR-124 overexpression, whereas it was blocked by LV-MALAT1 transfection. In vivo assays, overexpression of miR-124, or knockdown of MALAT1 exhibited beneficial effects on tumor weight, size, and volume, together with elevating the survival rate, tightly related with the progression of cervical cancer. In conclusion, LncRNA MALAT1 disabled the effects of miR-124 as an inhibitory sponge, accelerating the progression of cervical carcinoma.

The present study was performed to compare the increase in maternal serum concentrations of four placental proteins during the first half of 240 normal pregnancies. The proteins were pregnancy-associated plasma protein-A (PAPP-A), human chorionic gonadotrophin (HCG), human placental lactogen (HPL) hormone, and pregnancy-specific beta 1-glycoprotein (PSG), all produced by trophoblast cells. The median increases were observed to be very close to exponential growth curves. Based on simple assumptions, these growth curves could be explained as being solely dependent on the growth of the placenta. The assumptions were that the proteins were produced in the placenta at a constant rate per gram of placental cell mass and secreted into the circulation shortly after synthesis. Our investigations showed that for two of the proteins, PSG and HPL, the rate constants were, in fact, close to the reported growth rate of the placenta, whereas the PAPP-A production rate constant was significantly higher than those of the others. The production curve for HCG was very different from that of the other proteins. PAPP-A and HCG must therefore have more complicated mechanisms for regulating the production. An equation was constructed that permitted estimation of the molar production of the placental proteins per gram of placental cell mass per day during the first half of normal pregnancy. The value was highest for HPL and lowest for PAPP-A.

It has been firmly established that high-risk human papillomavirus (HPV) infection is associated with the appearance and persistence of anogenital dysplasia. However, only a small fraction of HPV-infected patients ever develop cervical cancer. Therefore, other factors must contribute for the development of a malignant phenotype in HPV-infected cells. Aberrant microRNA (miRNA) expression is nowadays recognized as a key factor for the development of several pathologies including cancer. Several studies on the miR-125a-5p function have suggested a tumor suppressor role in diverse cell types. Our previous results shown that miR-125a-5p induced cell-cycle arrest, proliferation inhibition and apoptosis in cervical carcinoma cells. Nevertheless, miR-125a-5p role and mRNA targets in cervical cancer are not fully established in cervical cancer. RT-qPCR analysis of miR-125a-5p expression showed differential expression on immortal and tumor cervical cell lines suggesting a role in malignant progression. To probe for possible miR-125a-5p targets we performed in silico analyses using several miRNA target analysis tools and validated the results with a panel of cervical immortal and tumor cell lines. The bio-informatics analysis showed Mark1, bcl-2 and vEGF genes as possible targets for miR-125a-5p. Further RT-qPCR and immunoblot analysis indicated an inverse correlation of miR125a-5p and MARK1 expression in tumor lines but not in immortal lines irrespective of the HPV content. Interestingly, transfection of a pre-miR-125a-5p mimic in C33-A cells (lacking HPV and with low endogenous miR-125a-5p levels), resulted in specific down-regulation of MARK1 and VEGF. Therefore, we suggest a potential role for miR-125a-5p in cervical carcinogenesis through the regulation of MARK1. Citation Format: Natalia Martinez Acuna, Maria L. Benitez Hess, Luis M. Alvarez Salas. Target mRNA analysis for miR-125a-5p in cervical carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4167. doi:10.1158/1538-7445.AM2013-4167

Functional differences among human cells have been difficult to identify by standard biochemical methods. Loss-of-function shRNA screens provide an unbiased method to compare protein requirements across cell lines. In previous work, we have studied kinase requirements in two settings, either among a panel of cells from numerous tissues or between two cell lines that differ only by the expression of a chosen oncoprotein or tumor suppressor protein. Here we examine the patterns of kinase requirements between two unrelated cells, the cervical carcinoma cell line HeLa and the renal carcinoma cell line 786-O. By using time courses of cell proliferation after shRNA transduction and by introducing different levels of the shRNAs, we were able to carefully compare the kinase requirements. These comparisons identified 10 kinases that were required in HeLa but not 786-O, and 5 kinases that were required in 786-O but not HeLa. The patterns of growth inhibition due to particular sets of shRNAs in a tumor cell line were shown to be similar in some but not all cell lines derived from the same tissue-specific cancer type. Differential kinase requirements promise to be useful in distinguishing important cell-to-cell functional variations and may lead to the identification of fingerprints for different physiological cell states.

The purpose of this study was to determine if squamous cell carcinoma antigen (SCCA), also known as SERPINB3, protects cervical cancer cells from ionizing radiation (IR)-induced death, and to determine the mechanism(s) of IR-induced cell death with or without SERPINB3. Cervical cancer remains a leading cause of cancer death in women worldwide despite advances in screening, vaccination and treatment. Recurrence after definitive chemoradiation occurs in up to 30-50% of patients with locally advanced disease. We and others have demonstrated that elevated serum SCCA portends poor prognosis in cervical cancer. In addition, CRISPR-Cas9-mediated knock out of SERPINB3 significantly sensitizes cervical tumor cells to IR in vitro. We hypothesize that SERPINB3 promotes radioresistance by protecting cells against lysosome damage and lysosomal mediated necrosis. Two cervical cancer cell lines with high SERPINB3 expression were edited using CRISPR-Cas9 technology at the SERPINB3 locus resulting in knock out (KO). B3-KO cells were significantly more radiation sensitive compared to control cells at all doses and time points evaluated. Cell death morphology in the B3-KO cells was necrotic, with large cytoplasmic single membraned vesicles, many of which were ruptured, consistent with that seen in lysosome-mediated necrosis. Live-cell time-lapse imaging showed loss of lysosome integrity in the hours leading up to cell death (propidium iodide nuclear staining). Western blot analysis showed low levels of caspase-3 and caspase-7 cleavage only at high doses and late time points after IR, with no evidence of phospho-MLKL or phospo-RIPK3 (markers of necroptosis), or gasdermin-D cleavage (marker of pyroptosis). Necrostatin, ferrostatin, liproxstatin and YVAD-Cho had little to no effect on cell death following IR, while pan-caspase inhibitors decreased IR-induced cell death in both control and B3-KO cells, and E64D decreased IR-induced cell death in B3-KO cells to a greater degree than control cells. Additionally, cervical tumor cell lines that had no detectable expression of SERPINB3 were engineered to expressed high levels of SERPINB3 (B3-wt) or SERPINB3 containing the P14 mutation A351R (B3-P14m), which lacks protease inhibitory function. B3-wt expressing cells displayed increased radiation resistance in clonogenic survival assays and mouse tumor models. Growth rate and radiation response of B3-p14m expressing tumors was similar to vector control. These data suggest that SERPINB3 is an important mediator of radiation response in cervical cancer and protects cells by inhibiting cysteine protease-dependent cell death, likely via lysosome-mediated necrosis. These findings support SERPINB3 as a potential target for novel radiosensitizing therapies. Citation Format: Songyan Wang, Clifford Luke, Victoria Shi, Perry Grigsby, Gary Silverman, Stephanie Markovina. SERPINB3 promotes radiation resistance in cervical cancer by inhibiting lysosome-mediated cell death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2426.