An Explorative Study on Calcium Electroporation for Low-risk Basal Cell Carcinoma.

In electrochemotherapy, permeabilization of the cell membrane by electric pulses increases the anti-tumour effect of chemotherapeutics. In calcium electroporation, chemotherapy is replaced by calcium chloride with obvious benefits. This study explores the effect and underlying mechanisms of calcium electroporation on basal cell carcinomas using either high- or low-frequency electroporation. Low-risk primary basal cell carcinomas were treated in local anaesthesia with intratumoral calcium chloride followed by electroporation with high (167 kHz) or low (5 kHz) frequencies. Non-complete responders were retreated after 3 months. The primary endpoint was tumour response 3 months after last calcium electroporation. Plasma membrane calcium ATPase was examined in various cell lines as plasma membrane calcium ATPase levels have been associated with calcium electroporation efficacy. Twenty-two out of 25 included patients complete the study and 7 of these (32%) achieved complete response at 3 months with no difference in efficacy between high- and low-frequency pulses. High-frequency calcium electroporation was significantly less painful (p=0.03). Plasma membrane calcium ATPase was increased 16-32-fold in basal cell carcinoma cell lines compared with 4 other cancer cell lines. Calcium electroporation for low-risk basal cell carcinomas does not fulfil the requirements of a new dermatological basal cell carcinoma treatment but may be useful as adjuvant treatment to surgery in more advanced basal cell carcinomas. The elevated PMCA levels in basal cell carcinomas may contribute to low efficacy.

A family with ulcerative colitis maps to 7p21.1 and comprises a region with regulatory activity for the aryl hydrocarbon receptor gene.

We have mapped a locus on chromosome 7p22.3-7p15.3 spanning a 22.4 Mb region for ulcerative colitis (UC) by whole genome linkage analyses of a large Danish family. The family represent three generations with UC segregating as an autosomal dominant trait with variable expressivity. The whole-genome scan resulted in a logarithm of odds score (LOD score) of Z = 3.31, and a whole genome sequencing (WGS) of two affected excluded disease-causing mutations in the protein coding genes. Two rare heterozygote variants, rs182281985:G>A and rs541426369:G>A, both with low allele frequencies (MAF A:0.0001, gnomAD ver3.1.2), were found in clusters of ChiP-seq transcription factors binding sites close to the AHR (aryl hydrocarbon receptor) gene and the UC associated SNP rs1077773:G>A. Testing the two SNPs in a promoter reporter assay for regulatory activity revealed that rs182281985:G>A influenced the AHR promoter. These results suggest a regulatory region that include rs182281985:G>A close to the UC GWAS SNP rs1077773:G>A and further demonstrate evidence that the AHR gene on the 7p-tel region is a candidate susceptible gene for UC.

Colorectal cancer-associated SNP rs17042479 is involved in the regulation of NAF1 promoter activity.

A novel risk locus at 4q32.2, located between the Nuclear Assembly Factor 1 (NAF1) and Follistatin Like 5 (FSTL5) genes, was associated with increased risk of developing colorectal cancer (CRC), with SNP rs17042479 being the most associated. However, the link between CRC development and the risk locus at 4q32.2 is unknown. We investigated the promoter activity of NAF1 and FSTL5 and analyzed the risk locus at 4q32.2 as gene regulatory region. Our results showed that the activity of the FSTL5 promoter was low compared to the NAF1 promoter. Analyses of the NAF1 promoter in conjunction with the region containing the risk locus at 4q32.2 showed that the region functions as gene regulatory region with repressor activity on NAF1 promoter activity. The SNP rs17042479(G) increased the repressor effect of the region. CRC patients' biopsies were genotyped for SNP rs17042479(A/G), and NAF1 expression profiles were examined. We found an association between SNP rs17042479(G), cancer stage and tumor location. Additionally, patients with SNP rs17042479(G) showed lower NAF1 expression in comparison to patients with SNP rs17042479(A) in tumor tissue and the NAF1 expression in tumor tissue was lower compared to healthy tissue. The results in the study imply that reduced NAF1 expression in the tumor contribute to a more aggressive phenotype. Furthermore, this study suggests that the SNP rs17042479(G) change the expression of NAF1 and thereby increases the risk of developing CRC.

Establishment of a luciferase-based method for measuring cancer cell adhesion and proliferation.

BACKGROUND: Methods measuring cell proliferation and adhesion are widely used but each hold limitations. We, therefore, introduce novel methods for measuring cell proliferation and adhesion based on CRISPR-modified cancer cell lines secreting luciferase to the growth media. MATERIALS AND METHODS: Using CRISPR genome editing, we generated stable luciferase-secreting LS174T, HCT 116, Caco-2, and PANC-1 cell lines. The modified cells were seeded, and luciferase activity was measured in the media and compared to Coulter counter cell counts and iCELLigence impedance assay to evaluate the value of the secreted luciferase activities as a measurement for adhesion and proliferation. RESULTS: Our results demonstrate that luciferase secreted into the media can be used quantifying cell proliferation and adhesion. The adhesion luciferase assay and the iCELLigence impedance assay showed similar results with increased significant difference observed in the luciferase assays. The luciferase proliferation assay showed increased growth following increased serum concentrations in all cell lines vs. only two cell lines in the iCELLigence impedance assay. CONCLUSIONS: Our results show that the luciferase adhesion and proliferation assays are reliable methods for measuring adhesion and proliferation. The luciferase assays have advantages over existing assays as they are highly sensitive, easy to perform, non-invasive and suitable as high-throughput measurements.

Colonic Stent as Bridge to Surgery for Malignant Obstruction Induces Gene Expressional Changes Associated with a More Aggressive Tumor Phenotype.

BACKGROUND: Colonic stent is recommended as a bridge to elective surgery for malignant obstruction to improve short-term clinical outcomes for patients with colorectal cancer. However, since the oncological outcomes remain controversial, this study aimed to investigate the impact of self-expandable metallic stent (SEMS) on the tumor microenvironment. METHODS: Patients treated with colonic stent as a bridge to surgery from 2010 to 2015 were identified from hospital records. Tumor biopsies and resected tumor samples of the eligible patients were retrieved retrospectively. Gene expression analysis was performed using the NanoString nCounter PanCancer IO 360 gene expression panel. RESULTS: Of the 164 patients identified, this study included 21 who underwent colonic stent placement as a bridge to elective surgery. Gene expression analysis revealed 82 differentially expressed genes between pre- and post-intervention specimens, of which 72 were upregulated and 10 downregulated. Among the significantly upregulated genes, 46 are known to have protumor functions, of which 26 are specifically known to induce tumorigenic mechanisms such as proliferation, migration, invasion, angiogenesis, and inflammation. In addition, ten differentially expressed genes were identified that are known to promote antitumor functions. CONCLUSION: SEMS induces gene expressional changes in the tumor microenvironment that are associated with tumor progression in colorectal cancer and may potentiate a more aggressive phenotype. Future studies are warranted to establish optimal timing of surgery after SEMS insertion in patients with obstructive colorectal cancer.

A novel approach for microRNA in situ hybridization using locked nucleic acid probes.

Identification of target tissue microRNAs (miR) using in situ hybridization (ISH), with digoxigenin-labeled locked nucleic acid (LNA) probes, is influenced by preanalytic parameters. To determine the best retrieval method for common microRNAs, a multiblock composed of paraffin-embedded tonsil, cervix, placenta, and hyperplastic prostate tissue were included. Tissue were fixed in 10% formalin in a range of 5-144 hours (h). Cut sections (5 µm) from the multiblock were subjected to combinations of pretreatment procedures: variable periods of proteinase K (PK) digestion or Heat-induced microRNA Retrieval (HmiRR) using target retrieval solution (TRS) pH 6.1 or 9, with or without enzymatic treatment (pepsin). Results for the overall categories: TRS pH 9 versus PK; p = 2.9e-23, TRS pH 9 versus TRS pH 6.1; p = 1.1e-14, TRS pH 6.1 versus PK; p = 2.9e-03. A long fixation time, resulted in the best microRNA preservation and staining intensity (long vs. short: p = 3.5e-47, long vs. moderate: p = 1.6e-44, moderate vs. short: p = 4.3e-16), was enhanced using HmiRR TRS pH 9 with or without pepsin providing high sensitivity and specificity. These observations conflict with other ISH techniques (e.g., messenger ribonucleic acid), which typically require shorter fixation periods, and therefore, further studies are warranted.

A transcriptomic overview of lung and liver changes one day after pulmonary exposure to graphene and graphene oxide.

Hazard evaluation of graphene-based materials (GBM) is still in its early stage and it is slowed by their large diversity in the physicochemical properties. This study explores transcriptomic differences in the lung and liver after pulmonary exposure to two GBM with similar physical properties, but different surface chemistry. Female C57BL/6 mice were exposed by a single intratracheal instillation of 0, 18, 54 or 162 µg/mouse of graphene oxide (GO) or reduced graphene oxide (rGO). Pulmonary and hepatic changes in the transcriptome were profiled to identify commonly and uniquely perturbed functions and pathways by GO and rGO. These changes were then related to previously analyzed toxicity endpoints. GO exposure induced more differentially expressed genes, affected more functions, and perturbed more pathways compared to rGO, both in lung and liver tissues. The largest differences were observed for the pulmonary innate immune response and acute phase response, and for hepatic lipid homeostasis, which were strongly induced after GO exposure. These changes collective indicate a potential for atherosclerotic changes after GO, but not rGO exposure. As GO and rGO are physically similar, the higher level of hydroxyl groups on the surface of GO is likely the main reason for the observed differences. GO exposure also uniquely induced changes in the transcriptome related to fibrosis, whereas both GBM induced similar changes related to Reactive Oxygen Species production and genotoxicity. The differences in transcriptomic responses between the two GBM types can be used to understand how physicochemical properties influence biological responses and enable hazard evaluation of GBM and hazard ranking of GO and rGO, both in relation to each other and to other nanomaterials.

CDX2 expression and perioperative patient serum affects the adhesion properties of cultured colon cancer cells.

BACKGROUND: Colon cancer is one of the most commonly diagnosed types of cancer with surgical resection of the tumor being the primary choice of treatment. However, the surgical stress response induced during treatment may be related to a higher risk of recurrence. The aim of this study was to examine the effect of surgery on adhesion of cultured colon cancer cells with or without expression of the tumour suppressor CDX2. METHOD: We enrolled 30 patients undergoing elective, curatively intended laparoscopic surgery for colon cancer in this study. Blood samples were drawn 1 day prior to surgery and 24 h after surgery. The samples of pre- and postoperative serum was applied to wild type colon cancer LS174T cells and CDX2 inducible LS174T cells and adhesion was measured with Real-Time Cell-Analysis iCELLigence using electrical impedance as a readout to monitor changes in the cellular adhesion. RESULTS: Adhesion abilities of wild type LS174T cells seeded in postoperative serum was significantly increased compared to cells seeded in preoperative serum. When seeding the CDX2 inducible LS174T cells without CDX2 expression in pre- and postoperative serum, no significant difference in adhesion was found. However, when inducing CDX2 expression in these cells, the adhesion abilities in pre- and postoperative serum resembled those of the LS174T wild type cell line. CONCLUSIONS: We found that the adhesion of colon cancer cells was significantly increased in postoperative versus preoperative serum, and that CDX2 expression affected the adhesive ability of cancer cells. The results of this study may help to elucidate the pro-metastatic mechanisms in the perioperative phase and the role of CDX2 in colon cancer metastasis.

HNF4α and CDX2 Regulate Intestinal YAP1 Promoter Activity.

The Hippo pathway is important for tissue homeostasis, regulation of organ size andgrowth in most tissues. The co-transcription factor yes-associated protein 1 (YAP1) serves as a maindownstream effector of the Hippo pathway and its dysregulation increases cancer development andblocks colonic tissue repair. Nevertheless, little is known about the transcriptional regulation ofYAP1 in intestinal cells. The aim of this study to identify gene control regions in the YAP1 gene andtranscription factors important for intestinal expression. Bioinformatic analysis of caudal typehomeobox 2 (CDX2) and hepatocyte nuclear factor 4 alpha (HNF4α) chromatin immunoprecipitatedDNA from differentiated Caco-2 cells revealed potential intragenic enhancers in the YAP1 gene.Transfection of luciferase-expressing YAP1 promoter-reporter constructs containing the potentialenhancer regions validated one potent enhancer of the YAP1 promoter activity in Caco-2 and T84cells. Two potential CDX2 and one HNF4α binding sites were identified in the enhancer by in silicotranscription factor binding site analysis and protein-DNA binding was confirmed in vitro usingelectrophoretic mobility shift assay. It was found by chromatin immunoprecipitation experimentsthat CDX2 and HNF4α bind to the YAP1 enhancer in Caco-2 cells. These results reveal a previouslyunknown enhancer of the YAP1 promoter activity in the YAP1 gene, with importance for highexpression levels in intestinal epithelial cells. Additionally, CDX2 and HNF4α binding areimportant for the YAP1 enhancer activity in intestinal epithelial cells.

Rectal Insulin Instillation Inhibits Inflammation and Tumor Development in Chemically Induced Colitis.

BACKGROUND AND AIMS: Epithelial expression of the insulin receptor in the colon has previously been reported to correlate with extent of colonic inflammation. However, the impact of insulin signalling in the intestinal mucosa is still unknown. Here, we investigated the effects of inactivating the epithelial insulin receptor in the intestinal tract, in an experimental model of inflammation-induced colorectal cancer. METHODS: The mice were generated by utilizing the intestinal- and epithelial-specific villin promoter and the Cre-Lox technology. All mice included in the cohorts were generated by crossing [vil-Cre-INSR+/-] × [INSRfl/fl] to obtain [vil-Cre-INSR-/-], and their floxed littermates [INSRfl/fl] served as the control group. For the intervention study, phosphate-buffered saline with or without insulin was instilled rectally in anaesthetized wild-type mice with chemically induced colitis. RESULTS: We found higher endoscopic colitis scores together with potentiated colonic tumorigenesis in the knockout mice. Furthermore, we showed that topically administered insulin in inflamed colons of wild-type mice reduced inflammation-induced weight loss and improved remission in a dose-dependent manner. Mice receiving rectal insulin enemas exhibited lower colitis endoscopic scores and reduced cyclooxygenase 2 mRNA expression, and developed significantly fewer and smaller tumours compared with the control group receiving phosphate-buffered saline only. CONCLUSIONS: Rectal insulin therapy could potentially be a novel treatment, targeting the epithelial layer to enhance mucosal healing in ulcerated areas. Our findings open up new possibilities for combination treatments to synergize with the existing anti-inflammatory therapies.

Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide.

We investigated toxicity of 2-3 layered >1 µm sized graphene oxide (GO) and reduced graphene oxide (rGO) in mice following single intratracheal exposure with respect to pulmonary inflammation, acute phase response (biomarker for risk of cardiovascular disease) and genotoxicity. In addition, we assessed exposure levels of particulate matter emitted during production of graphene in a clean room and in a normal industrial environment using chemical vapour deposition. Toxicity was evaluated at day 1, 3, 28 and 90 days (18, 54 and 162 µg/mouse), except for GO exposed mice at day 28 and 90 where only the lowest dose was evaluated. GO induced a strong acute inflammatory response together with a pulmonary (Serum-Amyloid A, Saa3) and hepatic (Saa1) acute phase response. rGO induced less acute, but a constant and prolonged inflammation up to day 90. Lung histopathology showed particle agglomerates at day 90 without signs of fibrosis. In addition, DNA damage in BAL cells was observed across time points and doses for both GO and rGO. In conclusion, pulmonary exposure to GO and rGO induced inflammation, acute phase response and genotoxicity but no fibrosis.

In Vitro Functional Analyses of Infrequent Nucleotide Variants in the Lactase Enhancer Reveal Different Molecular Routes to Increased Lactase Promoter Activity and Lactase Persistence.

The genetic trait that allows intestinal lactase to persist into adulthood in some 35% of humans worldwide operates at the level of transcription, the effect being caused by cis-acting nucleotide changes upstream of the lactase gene (LCT). A single nucleotide substitution, -13910 C>T, the first causal variant to be identified, accounts for lactase persistence over most of Europe. Located in a region shown to have enhancer function in vitro, it causes increased activity of the LCT promoter in Caco-2 cells, and altered transcription factor binding. Three other variants in close proximity, -13907 C>G, -13915 T>C and -14010 G>C, were later shown to behave in a similar manner. Here, we study four further candidate functional variants. Two, -14009 T>G and -14011 C>T, adjacent to the well-studied -14010 G>C variant, also have a clear effect on promoter activity upregulation as assessed by transfection assays, but notably are involved in different molecular interactions. The results for the two other variants (-14028 T>C, -13779 G>C) were suggestive of function, -14028*C showing a clear change in transcription factor binding, but no obvious effect in transfections, while -13779*G showed greater effect in transfections but less on transcription factor binding. Each of the four variants arose on independent haplotypic backgrounds with different geographic distribution.

HOXB4 Gene Expression Is Regulated by CDX2 in Intestinal Epithelial Cells.

The mammalian Caudal-related homeobox transcription factor 2 (CDX2) plays a key role in the homeobox regulatory network and is essential in regulating the expression of several homeobox (HOX) genes during embryonic development, particularly in the gut. Genome-wide CDX2 chromatin immunoprecipitation analysis and expression data from Caco2 cells also suggests a role for CDX2 in the regulation of HOXB4 gene expression in the intestinal epithelium. Thus, the aim of this study was to investigate whether HOXB4 gene expression is regulated by CDX2 in the intestinal epithelium. We demonstrated binding of CDX2 to four different CDX2 binding sites in an enhancer region located upstream of the HOXB4 transcription start site. Mutations in the CDX2 binding sites reduced HOXB4 gene activity, and knock down of endogenous CDX2 expression by shRNA reduced HOXB4 gene expression. This is the first report demonstrating the CDX2 regulation of HOXB4 gene expression in the developed intestinal epithelium, indicating a possible role for HOXB4 in intestinal homeostasis.

The prognostic value of polycomb group protein B-cell-specific moloney murine leukemia virus insertion site 1 in stage II colon cancer patients.

The aim of this study was to investigate the prognostic value of B-cell-specific moloney murine leukemia virus insertion site 1 (BMI1) protein expression in primary tumors of stage II colon cancer patients. BMI1 protein expression was assessed by immunohistochemistry in a retrospective patient cohort consisting of 144 stage II colon cancer patients. BMI1 expression at the invasive front of the primary tumors correlated with mismatch repair status of the tumors. Furthermore, BMI1 expression at the luminal surface correlated with T-stage, tumor location, and the histological subtypes of the tumors. In a univariate Cox proportional hazard analysis, no statistical significant association between risk of relapse and BMI1 protein expression at the invasive front (HR: 1.12; 95% CI 0.78-1.60; p = 0.53) or at the luminal surface of the tumor (HR: 1.06; 95% CI 0.75-1.48; p = 0.70) was found. Likewise, there was no association between 5-year overall survival and BMI1 expression at the invasive front (HR: 1.12; 95% CI 0.80-1.56; p = 0.46) or at the luminal surface of the tumor (HR: 1.16; 95% CI 0.86-1.60; p = 0.33). In conclusion, BMI1 expression in primary tumors of stage II colon cancer patients could not predict relapse or overall survival of the patients, thus having a limited prognostic value in stage II colon cancer patients.

SOX9 expression predicts relapse of stage II colon cancer patients.

The aim of this study was to investigate if the protein expression of sex-determining region y-box 9 (SOX9) in primary tumors could predict relapse of stage II colon cancer patients. One hundred forty-four patients with stage II primary colon cancer were retrospectively enrolled in the study. SOX9 expression was evaluated by immunohistochemistry, and mismatch repair status was assessed by both immunohistochemistry and promoter hypermethylation assay. High SOX9 expression at the invasive front was significantly associated with lower risk of relapse when including the SOX9 expression as a continuous variable (from low to high expression) in univariate (hazard ratio [HR], 0.73; 95% confidence interval [CI], 0.56-0.94; P = .01) and multivariate Cox proportional hazards analyses (HR, 0.75; 95% CI, 0.58-0.96; P = .02), adjusting for mismatch repair deficiency and histopathologic risk factors. Conversely, low SOX9 expression at the invasive front was significantly associated with high risk of relapse, when including SOX9 expression as a dichotomous variable, in univariate (HR, 2.32; 95% CI, 1.14-4.69; P = .02) and multivariate analyses (HR, 2.32; 95% CI, 1.14-4.69; P = .02), adjusting for histopathologic risk factors and mismatch repair deficiency. In conclusion, high levels of SOX9 of primary stage II colon tumors predict low risk of relapse, whereas low levels of SOX9 predict high risk of relapse. SOX9 may have an important value as a biomarker when evaluating risk of relapse for personalized treatment.

Impact of in Vivo Ischemic Time on RNA Quality--Experiences from a Colon Cancer Biobank.

Considerable effort has been made to improve differentiated diagnostics as well as personalized treatment for colorectal cancer patients. High-quality fresh frozen tissue is often required to investigate relevant molecular signatures in these patients. In RNA expression studies, the "RNA integrity number" is widely accepted as a reliable marker of RNA quality. Here, we investigate the feasibility of obtaining high-quality tissue from a colon cancer biobank and the impact of in vivo ischemic time and various technical and clinicopathological factors on RNA quality. Biopsies were obtained immediately following the tumor removal. The time from clamping the main arterial supply to resection and removal of the tumor was used to estimate the in vivo ischemic time. We did not observe a significant difference in RNA quality between normal tissue and tumor tissue. We observed a significant correlation between in vivo ischemic time and RNA quality in normal tissue (r = -0.24, p<0.001) but not in tumor tissue. Male gender and laparoscopic procedure were also significantly associated with lower RNA quality in normal tissue only. In tumor tissue, poor differentiation was associated with low RNA quality. In conclusion, in vivo ischemic time, surgical procedure, and gender have minor but significant effects on the quality of RNA from normal colon tissue but not tumor tissue. Poorly differentiated tumors are associated with lower RNA quality. Although its impact is low, it can still be considered to note in vivo ischemic time in colon cancer specimen procurement.

High interleukin-6 mRNA expression is a predictor of relapse in colon cancer.

AIM: To investigate the expression of interleukin-6 (IL6) in colon cancer tissue, and to examine if the risk of relapse is influenced by IL6 expression. MATERIALS AND METHODS: Fresh-frozen biopsies from tumor and normal adjacent tissues were taken from patients with colon cancer during surgery and stored at -80 °C. mRNA expression for interleukin-6 was evaluated with reverse transcription real time quantitative polymerase chain reaction. Survival analyses were carried-out using a Cox competing risk regression model. RESULTS: IL6 mRNA was significantly more highly expressed in tumor tissue compared to normal adjacent tissue (p<0.001). We found no significant association with regard to IL6 expression and histological differentiation or cancer stage. We found a significant association between high IL6 expression and risk of relapse (Hazard Ratio=2.23, 95% CI=1.10-4.53; p<0.05), also when adjusted for clinicopathological characteristics (Hazard Ratio=2.16, 95% CI=1.07-4.40; p<0.05). CONCLUSION: Interleukin-6 is up-regulated in colon cancer tissue at the transcriptional level and is significantly associated with increased risk of relapse.

Clinical implications of intestinal stem cell markers in colorectal cancer.

Colorectal cancer (CRC) still has one of the highest incidence and mortality rate among cancers. Therefore, improved differential diagnostics and personalized treatment are still needed. Several intestinal stem cell markers have been found to be associated with CRC and might have a prognostic and predictive significance in CRC patients. This review provides an overview of the intestinal stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), B cell-specific Moloney murine leukemia virus insertion site 1 (BMI1), Musashi1 (MSI1), and sex-determining region y-box 9 (SOX9) and their implications in human CRC. The exact roles of the intestinal stem cell markers in CRC development and progression remain unclear; however, high expression of these stem cell markers have a potential prognostic significance and might be implicated in chemotherapy resistance.

Transcriptional control of steroid biosynthesis genes in the Drosophila prothoracic gland by ventral veins lacking and knirps.

Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.