**1. Introduction**

Prostate cancer (PCa) is the second most common cancer type in men and the sixth leading cause of cancer-related death in men worldwide [1]. The discovery of new therapeutic avenues in PCa, and the development of effective drugs in the era of personalized medicine, would greatly benefit from the field of proteomics. The proteomics approach for a high-throughput study of biological samples by mass spectrometry has emerged as one of the main analytical strategies from the last years, and proteomic-based studies have greatly improved cancer research. Thus, proteomics represents an important tool for the identification of new molecular targets for PCa's tailored therapy.

**Citation:** Lage-Vickers, S.; Sanchis, P.; Bizzotto, J.; Toro, A.; Sabater, A.; Lavignolle, R.; Anselmino, N.; Labanca, E.; Paez, A.; Navone, N.; et al. Exploiting Interdata Relationships in Prostate Cancer Proteomes: Clinical Significance of HO-1 Interactors. *Antioxidants* **2022**, *11*, 290. https://doi.org/10.3390/ antiox11020290

Academic Editors: Elias Lianos, Maria G. Detsika and Alessandra Napolitano

Received: 6 December 2021 Accepted: 27 January 2022 Published: 31 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Inflammation is widely recognized as a hallmark of cancer [2]. Cell proliferation is enhanced in an inflammatory microenvironment rich in cytokines, growth factors, and agents that cause DNA damage [3]. This combination of factors makes the risk of developing a tumor much higher [3]. Further, inflammatory cells release reactive oxygen species (ROS), which generate oxidative stress and damage the DNA of neighboring epithelial cells, thus accelerating the evolution towards a more malignant phenotype [4]. Elevated intracellular ROS levels might affect several signaling pathways, resulting in the activation or repression of processes related to cell proliferation, motility, and survival [5,6]. Although the cause of prostatic inflammation is uncertain in most cases, it is believed that viral or bacterial infections, physical trauma from urinary reflux, dietary factors, estrogens, or a combination of these factors, could contribute to the establishment of an inflammatory microenvironment [7].

Heme oxygenase 1 (HO-1), the rate-limiting enzyme that catalyzes heme degradation, is a key player in cellular responses to pro-oxidative and pro-inflammatory insults [8,9]. HO-1 participates in cell homeostasis by attenuating inflammation, reducing oxidative injury, and regulating cell proliferation [10]. Reports from our laboratory documented that HO-1 has a strong anti-tumoral effect in vivo and in vitro in PCa [8,11–16]. Moreover, HO-1 induction in PCa cells impairs cell proliferation, invasion, and migration in vitro, and angiogenesis and tumor growth in vivo [11,12]. HO-1 is recognized as an integral smooth endoplasmic reticulum membrane protein; however, it has been detected in other subcellular compartments, including the nucleus [13,17,18]. It has been suggested that HO-1 undergoes proteolytic degradation at its carboxy-terminal hydrophobic end, which would facilitate its entry into the nucleus [13]. Interestingly, this truncated form of HO-1 does not possess catalytic activity [17]. It has been proposed that HO-1 has a non-canonical function in the nucleus, participating in the regulation of the activity of nuclear transcription factors and even regulating its own expression [19]. In line with this, we have previously documented that HO-1 binds to the proximal promoter of genes involved in PCa, such as the prostate-specific antigen (PSA), and represses androgen receptor (AR) activation revealing an undescribed function for HO-1 in the nucleus [12].

Due to the pleiotropic actions of HO-1, we hypothesized that its multiple functions could be mediated by interactions with several other relevant proteins associated with the carcinogenic process. Through co-immunoprecipitation assays, we previously verified that HO-1 interacts with STAT3, producing its retention in the cytoplasm of PCa cells [13]. Another HO-1 interactor protein in PCa cells identified by our group was Annexin A2 (ANXA2) [20], a key molecule in the adhesion process of PCa cells to the bone microenvironment. We found that HO-1 modulation in tumor cells interferes with ANXA2-mediated signaling [20]. These results clearly suggest that HO-1 is involved in cellular processes beyond the degradation of the heme group. However, further research into the mechanisms associated with HO-1 non-canonical functions is needed. Given that HO-1 does not show DNA binding motifs, it is possible that HO-1 needs to interact with transcription factors to fulfill its regulatory function in the cell nuclei.

To further our analysis, in this work, we undertook a proteomics approach to assess whether in PCa cells and under oxidative stress conditions, HO-1 could interact with proteins previously documented to have nuclear localization. Further, we evaluated the clinical relevance of such a network in PCa patients and performed correlation analyses among HO-1 and its partners, selecting those with higher correlation and building a risk score model. Taking into account all of our results, we report novel interactions between HO-1 and HSPB1, DDB1, and 14-3-3ζ/δ, highlighting their clinical relevance in PCa.

#### **2. Materials and Methods**

#### *2.1. Cell Culture, Treatments, Reagents, and Antibodies*

PC3 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were routinely cultured in RPMI 1640 (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Internegocios, Mercedes, Buenos Aires, Argentina), penicillin 100 U/mL, streptomycin 100 μg/mL, and amphotericin 0.5 μg/mL. Cells were cultured at 37 ◦C and 5% CO2.

For PC3 H2O2 treatment, cells were treated with 200 μM for 30 min, prepared in sterile PBS 1X. After treatment, cells were incubated in complete medium for 24 h and were harvested for the different experiments performed.

#### *2.2. Antibodies*

Monoclonal rabbit anti-human HO-1, anti-human 14-3-3ζ/δ and anti-human IgG antibodies were obtained from Cell Signaling (Danvers, MA, USA). Monoclonal mouse anti-human HO-1 antibody was obtained from Abcam (Cambridge, UK). Horseradish peroxidase (HRP) conjugated secondary anti-mouse antibody was obtained from Cell Signaling (Danvers, MA, USA). Secondary antibodies associated with the Alexa 555 and Alexa 647 fluorophores were obtained from Molecular Probes, Invitrogen (Carlsbad, CA, USA).

#### *2.3. PEBG-GST-HO-1 Cloning*

The vector pEBG-GST-HO-1 was generated by cloning the copy DNA sequence (cDNA) encoding the human HO-1 gene (*HMOX1*) into the restriction sites *BamHI* and *NotI* of the mammalian expression vector pEBG-GST (Addgene, Watertown, MA, USA). This strategy results in the fusion of the GST peptide at the N-terminus of HO-1. The sequences of primers used were: forward—5 -GCCGGATCCATGGAGCGTCCGCAAC-3 ; reverse—5 - GCCGCGGCCGCCATTCACATGGCATAAAGC-3 .
