Photodynamic therapy (PDT) is a valuable treatment method for vulvar intraepithelial neoplasia (VIN). It allows for the treatment of a multifocal disease with minimal tissue destruction. 5-Aminolevulinic acid (5-ALA) is the most commonly used prodrug, which is converted in the heme pathway to protoporphyrin IX (PpIX), an actual photosensitizer (PS). Unfortunately, not all patients treated with PDT undergo complete remission. The main cause of their failure is resistance to anticancer therapy. In many cancers, resistance to various anticancer treatments is correlated with increased activity of the DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1). Enhanced activity of drug pumps may also affect the effectiveness of therapy. To investigate whether multidrug resistance mechanisms underlie PDT resistance in VIN, porphyrins were isolated from sensitive and resistant vulvar cancer cells and their culture media. APE1 activity was measured, and survival assay after PDT combined with APE1 inhibitor was performed. Our results revealed that resistant cells accumulated and effluxed less porphyrins than sensitive cells, and in response to PDT, resistant cells increased APE1 activity. Moreover, PDT combined with inhibition of APE1 significantly decreased the survival of PDT-resistant cells. This means that resistance to PDT in vulvar cancer may be the result of alterations in the heme synthesis pathway. Moreover, increased APE1 activity may be essential for the repair of PDT-mediated DNA damage, and inhibition of APE1 activity may increase the efficacy of PDT.

Acromegaly results from growth hormone hypersecretion, predominantly caused by a somatotroph pituitary neuroendocrine tumor (PitNET). Acromegaly-causing tumors are histologically diverse. Our aim was to determine transcriptomic profiles of various somatotroph PitNETs and to evaluate clinical implication of differential gene expression. A total of 48 tumors were subjected to RNA sequencing, while expression of selected genes was assessed in 134 tumors with qRT-PCR. Whole-transcriptome analysis revealed three transcriptomic groups of somatotroph PitNETs. They differ in expression of numerous genes including those involved in growth hormone secretion and known prognostic genes. Transcriptomic subgroups can be distinguished by determining the expression of marker genes. Analysis of the entire cohort of patients confirmed differences between molecular subtypes of tumors. Transcriptomic group 1 includes ~20% of acromegaly patients with GNAS mutations-negative, mainly densely granulated tumors that co-express GIPR and NR5A1 (SF-1). SF-1 expression was verified with immunohistochemistry. Transcriptomic group 2 tumors are the most common (46%) and include mainly GNAS-mutated, densely granulated somatotroph and mixed PitNETs. They have a smaller size and express favorable prognosis-related genes. Transcriptomic group 3 includes predominantly sparsely granulated somatotroph PitNETs with low GNAS mutations frequency causing ~35% of acromegaly. Ghrelin signaling is implicated in their pathogenesis. They have an unfavorable gene expression profile and higher invasive growth rate.

Corticotroph pituitary adenomas commonly cause Cushing’s disease (CD), but some of them are clinically silent. The reason why they do not cause endocrinological symptoms remains unclear. We used data from small RNA sequencing in adenomas causing CD (n = 28) and silent ones (n = 20) to explore the role of miRNA in hormone secretion and clinical status of the tumors. By comparing miRNA profiles, we identified 19 miRNAs differentially expressed in clinically functioning and silent corticotroph adenomas. The analysis of their putative target genes indicates a role of miRNAs in regulation of the corticosteroid receptors expression. Adenomas causing CD have higher expression of hsa-miR-124-3p and hsa-miR-135-5p and lower expression of their target genes NR3C1 and NR3C2. The role of hsa-miR-124-3p in the regulation of NR3C1 was further validated in vitro using AtT-20/D16v-F2 cells. The cells transfected with miR-124-3p mimics showed lower levels of glucocorticoid receptor expression than control cells while the interaction between miR-124-3p and NR3C1 3′ UTR was confirmed using luciferase reporter assay. The results indicate a relatively small difference in miRNA expression between clinically functioning and silent corticotroph pituitary adenomas. High expression of hsa-miR-124-3p in adenomas causing CD plays a role in the regulation of glucocorticoid receptor level and probably in reducing the effect of negative feedback mediated by corticosteroids.

Protein deubiquitinases USP8 and USP48 are known driver genes in corticotroph pituitary neuroendocrine tumors (PitNETs). USP8 mutations have pleiotropic effects that include notable changes in genes’ expression. Genes involved in cell cycle regulation were found differentially expressed in mutated and wild-type tumors. This study aimed to verify difference in the expression level of selected cell cycle-related genes and investigate their potential role in response to cell cycle inhibitors. Analysis of 70 corticotroph PitNETs showed that USP8-mutated tumors have lower CDKN1B, CDK6, CCND2 and higher CDC25A expression. USP48-mutated tumors have lower CDKN1B and CCND1 expression. A lower p27 protein level in mutated than in wild-type tumors was confirmed that may potentially influence the response to small molecule inhibitors targeting the cell cycle. We looked for the role of USP8 mutations or a changed p27 level in the response to palbociclib, flavopiridol and roscovitine in vitro using murine corticotroph AtT-20/D16v-F2 cells. The cells were sensitive to each agent and treatment influenced the expression of genes involved in cell cycle regulation. Overexpression of mutated Usp8 in the cells did not affect the expression of p27 nor the response to the inhibitors. Downregulating or upregulating p27 expression in AtT-20/D16v-F2 cells also did not affect treatment response.

ObjectivePituitary neuroendocrine corticotroph tumors commonly cause Cushing’s disease (CD) that results from increased adrenocorticotropic hormone (ACTH) secretion by the pituitary tumor and consequent increase of cortisol levels in blood. However, in some patients, corticotroph tumors remain clinically non-functioning. Cortisol secretion is regulated by the hypothalamic–pituitary–adrenal axis and includes a negative feedback between cortisol and ACTH secretion. Glucocorticoids reduce ACTH level both by hypothalamic regulation and acting on corticotrophs via glucocorticoid (GR) and mineralocorticoid (MR) receptors. The aim of the study was to determine the role of GR and MR expression at mRNA and protein levels in both functioning and silent corticotroph tumors.MethodsNinety-five patients were enrolled, including 70 with CD and 25 with silent corticotroph tumors. Gene expression levels of NR3C1 and NR3C2 coding for GR and MR, respectively, were determined with qRT-PCR in the two tumor types. GR and MR protein abundance was assessed with immunohistochemistry.ResultsBoth GR and MR were expressed in corticotroph tumors. Correlation between NR3C1 and NR3C2 expression levels was observed. NR3C1 expression was higher in silent than in functioning tumors. In CD patients NR3C1 and NR3C2 levels were negatively correlated with morning plasma ACTH levels and tumor size. Higher NR3C2 was confirmed in patients with remission after surgery and in densely granulated tumors. Expression of both genes and GR protein was higher in USP8-mutated tumors. Similar relationship between USP8 mutations and expression levels were observed in analysis of silent tumors that also revealed a negative correlation between GR and tumor size and higher NR3C1 expression in densely granulated tumors.ConclusionsAlthough the associations between gene/protein expression and patients clinical features are not strong, they consistently show an evident trend in which higher receptor expression corresponds to more favorable clinical characteristics.

Photodynamic therapy (PDT) is a low-invasive treatment method that can be used to treat VIN patients. A photosensitizer (PS) applied to a patient is activated with use of the appropriate wavelength of light, which in an oxygen environment leads to the formation of a reactive oxygen species (ROS) that destroys the tumor. However, cells can protect themselves against these cytotoxic products by increasing their antioxidant mechanisms and repair capacity. Changes in the cytoskeleton may also influence resistance to PDT. Our results revealed that PDT-resistant cells changed the amount of ROS. Cells resistant to PDT A-431 exhibited a decreased ROS level and showed higher viability after oxidizing agent treatment. Resistant Cal-39 cells exhibited a decreased O2− level but increased other ROS. This provides protection from PDT but not from other oxidizing agents. Moreover, PDT leads to alterations in the cytoskeleton that may result in an epithelial-mesenchymal transition (EMT) or increased adhesion. Both EMT and cell adhesion may activate signaling pathways involved in survival. This means that resistance to PDT in vulvar cancer may be at least in part a result of changes in ROS level and alterations in the cytoskeleton.

Acromegaly results from growth hormone hypersecretion caused by somatotroph pituitary neuroendocrine tumor (PitNET). Our molecular profiling revealed that acromegaly-causing tumors form three distinct transcriptomic subgroups with different histological/clinical features. Transcriptomic subtypes of somatotroph tumors differ in the expression levels of numerous genes including those involved in hormone secretion and genes with known prognostic value. They can be distinguished by determining the expression of marker genes. Transcriptomic group 1 includes ~20% of acromegaly patients with GNAS mutations-negative, mainly densely granulated tumors with NR5A1 (SF-1) and GIPR co-expression. Group 2 tumors are the most common (46%) and include mainly GNAS-mutated, densely granulated somatotroph and mixed PitNETs. They have significantly smaller size and express favorable prognosis-related genes. Group 3 includes predominantly sparsely granulated somatotroph PitNETs with low GNAS mutations frequency causing ~35% of acromegaly cases. Ghrelin signaling is implied in their pathogenic mechanism, they have unfavorable gene expression profile, and invasive growth rate. Since a subgroup of somatotroph tumors have high NR5A1 expression, using SF-1 as classification marker specific to gonadotroph PitNETs could be reconsidered.

Terapia fotodynamiczna (ang. photodynamic therapy, PDT) jest jedną z najmniej toksycznych metod walki z rakiem. Podany pacjentowi fotouczulacz (ang. photosensitizer, PS) akumuluje się w tkance guza, gdzie pod wpływem światła o odpowiedniej długości fali i intensywności dochodzi do jego aktywacji. Aktywowany PS w obecności tlenu prowadzi do powstania reaktywnych form tlenu (ang. reactive oxygen species, ROS), które powodują liczne uszkodzenia.  Powstałe nieodwracalne uszkodzenia prowadzą do śmierci komórek poprzez mechanizm apoptozy, nekrozy lub autofagii. Ponadto w wyniku PDT dochodzi do zapoczątkowania ostrej lokalnej reakcji zapalnej, która bierze udział w usuwaniu martwych komórek, przywróceniu normalnej homeostazy tkanek, a czasami i w rozwinięciu odporności ogólnoustrojowej. Jednak część komórek nowotworowych może przeżyć terapię i zmienić swój fenotyp, prowadząc do pojawienia się oporności. W ochronę przed cytotoksycznym efektem PDT mogą być zaangażowane mechanizmy prowadzące do obniżenia poziomu fotouczulacza w komórkach, zwiększone zdolności antyoksydacyjne, nadekspresja białek opiekuńczych czy aktywacja ścieżek sygnałowych sprzyjających przeżyciu.