Adrenocorticotropic hormone (ACTH)-secreting pituitary tumors that cause Cushing’s disease account for 5% to 10% of pituitary adenomas. Silent corticotroph adenomas are an uncommon subgroup of nonfunctioning pituitary tumors (NFPAs) (1) characterized by no biochemical or clinical evidence of Cushing’s disease (2, 3). They are generally diagnosed after surgery by positive immunostaining for ACTH (4). They represent approximately 20% of all corticotroph adenomas and approximately 5% of NFPAs (3, 5).

These silent pituitary tumors are usually macroadenomas and have a more aggressive behavior with a higher chance of hemorrhage and invasion of anatomical critical structures (6). On rare occasions, they transform to active Cushing’s disease, with high serum ACTH levels (5). Moreover, for silent corticotroph adenomas, there has been reported a high risk of recurrence. For those patients whose tumor recurs or progresses, treatment includes reoperation, combined medical approaches, and/or stereotactic radiotherapy (7–9). There is growing experience in the use of TMZ to treat these aggressive tumors and many studies that relate to its effectiveness with the expression of MGMT and other methyltransferases (10, 11).

The complexity of silent corticotroph tumors challenges their appropriate diagnosis and clinical management. Identification of markers that can predict the aggressive progression of pituitary tumors may be useful in the treatment strategy and could be of assistance in preventing recurrence.

β-Catenin is essential for embryonic development and is required for cell renewal/regeneration in adult life. β-Catenin-mediated signaling has been associated with tumor-initiating cells in multiple malignancies (12). Its activation promotes cell proliferation and supports tumor growth. Aberrant expression of β-Catenin has a role in cellular transformation, tissue invasion, and metastasis (13). Moreover, β-Catenin contributes to the protumoral angiogenic process in different cancers (14–16).

The present case is a rare silent corticotroph tumor that evolved into an aggressive ACTH secreting tumor and required repeated surgeries as well as combined medical treatment including Temozolomide (TMZ). Across the follow-up, pathology studies showed cellular abnormalities, increased Ki-67 proliferation index, variable p53 immunostaining, and necrotic areas. Molecular biology analysis showed singular changes in tumor vascularity as well as in the expression and localization of the β-CATENIN protein.

Our analysis and results contribute to understanding the processes that occur along different transforming stages in the aggressive pituitary tumor evolution. They provide promising data that could be helpful in new treatment design for aggressive and resistant pituitary tumor management.

Corticotroph tumors represent the predominant tumor subtype in cohorts of aggressive pituitary tumors and pituitary carcinomas (18). Moreover, it is known that silent corticotroph tumors may turn into aggressive tumors or carcinomas (19, 20). The peculiarities of silent corticotroph adenomas such as their high frequency of recurrence and their invasive capability led the research community to delve into the causes that make them such complex entities to handle (5, 8). Despite the multiple efforts, many questions remain unsolved, as to what mechanisms are driving their “silence” in the clinic, what turns them hormonally active in some cases, which cell markers could predict their evolution to aggressive behavior, or which could be indicative of a better response to treatments, among others.

Here we present a rare case of a patient with a silent corticotroph tumor that compromised the optic chiasm, which recurred as a giant, aggressive ACTH-secreting tumor with a high proliferation index and drug resistance. We describe the course of the lesion and analyzed the histopathological changes in samples of the initial, the relapsed, and the re-grown tumors.

During its transformation, the tumor turned from clinically silent (with no signs of Cushing disease) with a 1% Ki-67 proliferation index in 2008, to Cushing’s disease caused by a giant tumor, with high proliferation rates (9% and 4% in 2014 and 2015, respectively). Contrarily to this case, Moshkin et al. described a silent corticotroph tumor, which also turned recurrent and resisted conventional therapies, with no Ki-67 increments during tumor transformation (21).

The secreting tumor we present here expressed p53 and showed an apparent resistance to 3 months of TMZ treatment in 2014 and 2015 as it was already reported in the literature for ACTH secreting tumors (22).

Several markers, including different cell signaling components, have been investigated to reveal their implication in functioning and/or silent corticotroph pituitary adenomas (23–25). Aberrant activation of Wnt/β-Catenin signaling has been reported in many types of cancers (12, 26–29) as well as in pituitary tumors, in which both canonical and non-canonical Wnt pathways have been linked to tumor development (29–32). Moreover, mutations in the third exon of the β-Catenin gene (CTNNB1), which encodes the GSK3-β binding site and is the degradation-targeting box of β-Catenin, are infrequent in pituitary tumors (33, 34). Importantly, the knockdown of β-Catenin inhibited pituitary adenoma cell proliferation and migration probably by inhibiting AKT, STAT3, Cyclin D1, CDK4, MMP-2, and MMP-9. It suggests that β-Catenin may regulate various downstream molecules to enhance pituitary tumor cell proliferation and invasion (35).

Additionally, Wnt pathway inhibitor WIF-1 was found underexpressed in human bromocriptine-resistant prolactinomas (36) as well as in non-functioning pituitary tumors (37).

β-Catenin is known to play a role in cell-to-cell junctions by associating with α-catenin and E-cadherin in the membrane. Several mediators were described to dissociate β-catenin from adherens junctions and induce its translocation into the nucleus and activation of Wnt target genes (38, 39).

In our previous work in a cohort of human resistant prolactinomas, we described β-CATENIN expression that localized in membrane, cytoplasm, and nucleus of prolactinoma cells, and we showed a markedly decrease of this key protein in membranes when compared with normal pituitaries, which would indicate its activation (40).

Nevertheless, if we focus the search on the particular group of silent and aggressive corticotroph tumors, available data related to Wnt components’ expression or β-CATENIN relocalization are scarce (25). Indeed, molecular studies on different stages of this kind of pituitary tumor transformation are not commonly found in the literature.

In the current study, we quantified β-CATENIN expression in the samples from 2014 and 2015, which suggested, as we have already reported for resistant prolactinomas (40, 41), a β-CATENIN activation in corticotroph tumor aggressiveness. In this sense, Semba and colleagues also showed nuclear localization of β-CATENIN in half of the pituitary samples of the cohort they analyzed, which included a silent corticotroph adenoma. Normal glands in turn, only showed membrane β-CATENIN expression in that cohort (42).

Additionally to β-CATENIN relocalization, our present results suggest a remodeling of the vasculature across the aggressive transformation. Angiogenesis, a widely studied process by us and by other authors in pituitary pathogenesis (17, 43–48), can affect tumor promotion and therapy responses. Markers of angiogenesis as VEGF and vascular density were reported to increase in aggressive pituitary tumors, although their significance related to response to anti-angiogenic therapy is uncertain (49). In our present study, the evaluation of angiogenic parameters showed that the lesion evolved from a highly vascularized and less proliferative to an aggressive and less vascularized tumor, denoting that aggressiveness was driving tumor growth despite less vascularization. Contrarily, it was reported that microvascular density and VEGF expression were higher in pituitary carcinomas than in benign adenomas (50). In lactotroph tumors, indeed, tumor aggressiveness has been associated with neoangiogenesis, and with Endocan and VEGF overexpression among other factors (51). Similarly, in the case presented by Moshkin et al, with several surgeries as our case, a markedly vascular tumor with dilated, irregular capillaries and moderate VEGF immunostaining in almost all tumor cells was described. Nevertheless, other authors published a reduction in the microvascular density of NF and GH-secreting tumors (52) as well as in some pituitary tumor groups which included ACTH secreting tumors when compared to normal pituitaries (53). Potentially, the reduction in the vascularization that accompanied this tumor transformation could be the result of the fibrotic process found during the second surgery (clinical presentation) and may be mediated by TGF-β1, a stromal fibroblast activator and inhibitor of angiogenesis (54). Additionally, prolonged cabergoline treatment, known to induce fibrosis in tumors and decrease angiogenesis (45), might have also contributed to vascular area reduction (55).

It has been suggested that type I silent corticotroph adenomas have lower levels of MGMT than clinically active ACTH-secreting tumors (56), then, they could be good candidates for TMZ treatment. Contrarily, several works revealed a lack in the response to the drug in this type of pituitary tumor and in more aggressive cases (22, 57). According to a meta-analysis of all reported cases until December 2020, it was stated that functioning tumors respond better to TMZ than non-functioning ones (58).

Our patient received TMZ together with Cabergoline and Ketoconazole. This combination might have attenuated Cushing´s disease, stabilized tumor secretion, and partially decreased cell proliferation index from 9 to 4%, but it did not reduce tumor growth completely. To the contrary, the tumor regrew later. Given the undetectable levels of expression of MGMT in the ACTH secreting tumor (evaluated in 2014 and 2015 lesions), MGMT would not contribute to TMZ resistance and it would probably be the short TMZ cycle (only 3 months) the key point of tumor regrowth from the highly proliferative remnant.

Interestingly, Wnt/β-Catenin has been implicated in the chemoresistance of different types of cancer (15, 59, 60). In particular, its inhibition augments the cytotoxicity of the alkylating agent Temozolomide (TMZ) in colon carcinoma, glioblastoma, medulloblastoma, and neuroblastoma cell lines (61). Nevertheless, as far as we are concerned, the role of β-Catenin in silent-to-aggressive pituitary tumor progression under TMZ treatment has not been investigated yet. In the light of the present case, it seems that β-Catenin activation can contribute to pituitary tumor resistance to TMZ.

We could hypothesize that the activation and persistence of β-CATENIN we found herein is driving the pro tumoral processes, such as proliferation probably mediated by Cyclin D1 (recently demonstrated by us in in vitro models of prolactinomas 40), as well as disorganization of tumor vasculature and the increase in hormonal secretion. In turn, it could be indicating, that β-CATENIN activation might be driving tumor TMZ resistance.

Finally, in vivo and in vitro studies that explain Wnt signaling and active β-CATENIN participation in aggressive corticotroph tumor progression as well as the response to treatments could provide complementary information to consider Wnt signaling as a possible new therapeutic target for aggressive ACTH producing pituitary tumors.

The datasets for this article are not publicly available due to concerns regarding participant/patient anonymity. Requests to access the datasets should be directed to the corresponding author.

The study was approved by Comité de Ética en la Investigación y en uso de animales de experimentación de la Universidad Nacional del Noroeste de la Provincia de Buenos Aires (COENOBA). The patient provided the written informed consent to participate in the study.

GD performed experimental analyses, organized data, and wrote the first manuscript. SP contributed with experimental data quantification. GER, CDB, JPC and GS provided clinical data, imaging, and important clinical expertise. SB managed the case, performed the surgeries, highlighted the particularity of the case, and gave invaluable clinical information. CC organized the group and data, designed the work, and wrote the final version of the paper. All authors contributed to manuscript construction and revision and approved the final manuscript.

We thank the following institutions that financially supported our work, Agencia Nacional de Promoción Científica y Tecnológica, Argentina [PICT 901-2013], Consejo Nacional de Investigaciones Científicas y Técnicas and Universidad Nacional del Noroeste de la Provincia de Buenos Aires [PIO CONICET-UNNOBA 2015-2016 15720150100010CO], and SIB UNNOBA [#0241/2017]. The three grants were awarded to CC.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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