Breast cancer is the leading cause of cancer-related mortality among women worldwide and the most prevalent malignancy in this population [1]. In 2022, approximately 2.3 million new cases were identified, according to data from the Global Cancer Observatory (GLOBOCAN) of the International Agency for Research on Cancer (IARC) [2]. The main risk factors for the development of breast cancer include a family history of the disease, mutations in the BRCA1 and BRCA2 genes, prolonged use of hormone therapy, and lifestyle factors [3]. In addition, several viral infections have been investigated as potential contributors to breast carcinogenesis, including Epstein–Barr virus (EBV), mouse mammary tumor virus (MMTV), and human papillomavirus (HPV) [3].

HPV is a double-stranded DNA virus comprising more than 200 types, which are classified as low- or high-risk according to their oncogenic potential [4]. Among the high-risk types, HPV16 is the most prevalent, accounting for approximately 89% of HPV-positive oropharyngeal cancers and 52% of cervical cancer cases [5–7]. Furthermore, most lesions classified as grade 3 epithelial neoplasia (CIN3), considered premalignant and more likely to progress to invasive cancer, are associated with infection by this viral genotype [5, 7, 8].

The first evidence of an association between HPV and breast cancer was reported in 1992, and since then, numerous studies have explored this relationship [9, 10]. Studies assessing HPV prevalence in breast tissue report a wide variation ranging from 0 to 86.2%, with a pooled prevalence of 23% for viral DNA positivity, with genotype 16 being the most prevalent in these tissues [11, 12]. More recently, a study by Sakhi et al. [13] detected HPV in breast carcinoma tissues, of which 65.4% were positive for HPV16. However, other studies have not detected the presence of HPV16 in breast cancer tissues, highlighting the variability in HPV detection rates and viral genotype distribution among different studies [13–16]. Women with HPV-positive tumors have an approximately 5.9-fold higher likelihood of presenting a worse prognosis in invasive ductal carcinoma compared with those with HPV-negative tumors [17]. Additionally, the presence of HPV in breast tissue has also been associated with more aggressive grade III tumors and high expression of Ki-67, a protein that reflects the tumor cell proliferation rate [13].

HPV can modulate multiple signaling pathways involved in inflammation, cell proliferation, and immune response [18]. This modulation constitutes a viral mechanism that contributes to the initiation of carcinogenesis [19]. In studies of HPV-positive head and neck squamous cell carcinoma, increased activation of the PI3K/AKT/mTOR pathway has been observed. This pathway regulates cell proliferation, survival, invasion, migration, apoptosis, glucose metabolism, and DNA repair, and its activation is higher in HPV-positive cases than in HPV-negative ones [20].

This pathway is activated by the epidermal growth factor receptor (EGFR), a transmembrane glycoprotein and member of the protein kinase superfamily [21, 22]. EGFR is a cell surface receptor that binds epidermal growth factor, triggering receptor dimerization and tyrosine autophosphorylation, ultimately promoting cell proliferation [22, 23]. Studies have shown that elevated EGFR expression is associated with poor prognosis in breast cancer, particularly in triple-negative and HER2-positive subtypes [24, 25]. The negative regulator of this pathway, phosphatase and tensin homolog (PTEN), functions as a phosphatase by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to phosphatidylinositol 4,5-bisphosphate (PIP2), thereby interrupting the signal that activates the serine/threonine kinase AKT [20, 26]. In the absence of PTEN, AKT and the mammalian target of rapamycin (mTOR) become hyperactivated, leading to uncontrolled cell growth and resistance to apoptosis [20].

The PI3K/AKT/mTOR pathway plays a central role in breast cancer by regulating cell growth, survival, and tumor progression. Consequently, it has emerged as a key therapeutic target in the management of this malignancy [27–29]. Inhibitors of PI3K, AKT, and mTOR have been investigated in the treatment of different molecular subtypes of breast cancer, with some already approved by the Food and Drug Administration (FDA), such as everolimus, an mTOR inhibitor, and alpelisib, a PI3K inhibitor, indicated for luminal-type cancers [30]. In this context, investigating how HPV influences this pathway—particularly through its oncoproteins E5, E6, and E7—may reveal mechanisms by which the virus contributes to breast carcinogenesis and help identify novel therapeutic approaches for HPV-associated breast neoplasms. Accordingly, the aim of the present study is to elucidate the role of HPV in modulating the PI3K/AKT/mTOR pathway in breast tumors, thereby contributing to a better understanding of the relationship between HPV and breast cancer.

To date, few studies have assessed the expression of viral oncogenes in breast cancer tissues, which serve as direct markers of HPV16 activity [36, 37]. A major challenge in this approach is the acquisition of high-quality RNA, particularly from FFPE tissues, which are more readily available for research than fresh specimens [38, 39]. In the present study, 92 FFPE breast tissue samples were analyzed. High-quality RNA was successfully isolated from only 48 of these samples, corroborating the challenges frequently reported in the literature for this type of analysis.

In our cohort of breast tumor samples, 77.08% were positive for HPV16. Among the HPV16-positive cases, 64.58% were classified as luminal A or luminal B molecular subtypes. The luminal subtype is the most prevalent among breast cancers and comprises a highly heterogeneous group in terms of prognosis and therapeutic response [40]. Luminal A breast cancer is characterized by low proliferative activity and a more favorable prognosis, whereas luminal B tumors exhibit higher proliferative rates, a more aggressive phenotype, and reduced post-recurrence survival [41, 42]. The high frequency of HPV16 may be associated with the widespread prevalence of this genotype in the general population, both in Brazil and globally. In Brazil, studies have shown that the prevalence of cervical HPV infection ranges from approximately 13.7% to 54.3%, with HPV16 representing the most prevalent high-risk genotype across all regions of the country [43, 44]. At the global level, high-risk HPV types represent a significant proportion of HPV-positive infections, accounting for 54.3%, although this prevalence may vary according to geographic region and the detection methods employed [45, 46]. Furthermore, other studies have reported consistent findings, demonstrating a higher prevalence of HPV16 in the luminal A and luminal B subtypes [47–49].

HPV oncogenes interact with multiple signaling pathways, thereby contributing to tumorigenesis [50]. As described in the literature, the E6 and E7 oncogenes interact with pRb and p53, respectively, facilitating cell cycle progression, whereas the E5 oncogene plays a key role in immune evasion [51, 52]. In contrast to the extensively studied E6 and E7 oncoproteins, which primarily deregulate cell cycle control and proliferation, the E5 oncogene is distinguished by its role in modulating growth factor receptor signaling and regulating the immune response [53]. The E5 oncogene interacts with the vacuolar H⁺-ATPase (V-ATPase), promoting EGFR activation by inhibiting EGF-induced endosomal acidification, thereby reducing receptor degradation. In addition, E5 downregulates the expression of human leukocyte antigen (HLA), contributing to immune evasion [50]. Previous studies have indicated that elevated levels of E5 can potentiate the activation of receptors such as the EGFR, triggering signaling cascades that promote cell proliferation and tumor progression [54–56].

The samples analyzed in this study exhibited higher relative expression of the E5 oncogene compared with E6 and E7. Furthermore, no significant difference in expression was observed between E6 and E7. Ren et al. [53] reported higher E5 expression in HPV-associated head and neck cancer samples compared to E6 and E7 levels. Similarly, Nascimento et al. [16] reported elevated E5 oncogene expression in HPV16-positive breast cancer samples; however, no comparison with E6 and E7 was performed. Collectively, these findings, together with the results presented here, suggest that the E5 oncogene may play a pivotal role in viral persistence in non-anogenital HPV-associated cancers, potentially contributing to the activation of non-canonical mechanisms involved in tumorigenesis.

E5 is known to disrupt the expression of major histocompatibility complex class I (MHC I), thereby deregulating the antigen presentation machinery and impairing the activation of CD8⁺ T lymphocytes [57]. Collectively, these interactions promote viral immune evasion and contribute to malignant cellular transformation. The expression of this oncogene in head and neck cancers has been associated with resistance to anti-PD-L1 immunotherapy, thereby impairing the generation of new T cells [58]. In this context, the data presented herein underscore the need for a more comprehensive investigation of E5 in breast cancer, particularly considering its overexpression and its potential impact on the response to immunotherapy used in the treatment of breast cancer, especially in the triple-negative subtype [59].

Breast carcinogenesis involves the dysregulation of cellular signaling pathways that drive proliferation and promote cell survival [60, 61]. Among the most frequently dysregulated pathways is the PI3K/AKT/mTOR signaling cascade, which is often upregulated in breast cancer and contributes to multiple processes essential for tumorigenesis [62, 63]. Uncontrolled activation of this pathway affects cellular metabolism, growth, and proliferation, as well as the regulation of apoptotic and angiogenic processes [64, 65]. In this study, we investigated for the first time the modulation of this pathway by HPV oncogenes in breast tumors, revealing key features that are discussed below.

Dysregulation of PI3K in breast cancer contributes to hyperactivation of this pathway, thereby increasing cell proliferation, promoting tumor progression, and driving therapeutic resistance [66]. Accordingly, PI3K inhibitors have been employed as a therapeutic strategy due to the high frequency of alterations in this pathway, including elevated expression and mutation rates in cancer [67]. Compounds such as GDC-0084 have demonstrated, in vitro, the ability to induce apoptosis and inhibit cell proliferation, suggesting potential therapeutic benefit [28]. Furthermore, a study by Liu et al. [68] demonstrated that HPV16-associated lung cancer cells exhibited higher expression levels of the viral oncogenes E6 and E7. Notably, increased expression of these genes was correlated with activation of the PI3K/AKT pathway, which regulates the activity of key transcription factors [68]. Additionally, a study in cervical carcinoma cells harboring the E6 oncogene reported increased levels of phosphorylated PI3K, suggesting that HPV infection may influence the regulation of this protein [69]. However, this association warrants further investigation to confirm its significance and elucidate the underlying mechanisms.

AKT plays a central role in regulating cell proliferation, survival, and metabolism [70, 71]. In breast carcinoma, AKT can be activated by signaling pathways involving EGFR and PI3K and is frequently overexpressed in conjunction with PI3K, making it a key target for the development of novel therapeutic strategies [72, 73]. The results presented here revealed increased PI3K expression in the HPV16-positive group; however, no significant difference in AKT expression levels was observed between HPV16-positive and HPV16-negative breast cancer groups. These findings suggest that AKT expression may be regulated by alternative signaling pathways and post-transcriptional mechanisms beyond PI3K, potentially explaining the lack of significant differences at the mRNA level. In this context, our results also demonstrated a positive correlation between E6 and AKT, supporting the hypothesis that the absence of significant differences in AKT expression may be related to the low levels of E6 expression observed in this study. Notably, E6 has been shown to promote AKT phosphorylation, thereby enhancing the activity of eIF4E, a key regulator of the translation of genes involved in cell cycle progression, cell growth, and angiogenesis [74].

The mTOR plays a central role in regulating cellular metabolism, protein synthesis and degradation, survival, proliferation, and migration [75, 76]. Dysregulated oncogenes, hyperactivation of growth factor signaling via the PI3K/AKT and RAS/RAF pathways, elevated amino acid availability, and increased levels of pro-inflammatory cytokines collectively contribute to mTOR-driven tumorigenesis [77–79]. This gene is frequently dysregulated in breast cancer, either through gain-of-function mutations or increased activity of upstream signaling pathways [80]. In the present study, we observed a significant decrease in mTOR expression in the HPV16-positive breast cancer group (p < 0.001). Although previous studies have reported an association between HPV oncoproteins, particularly E6, and increased mTOR levels—and our correlation analysis also identified a positive association [81–83]—our findings indicate a reduction in mTOR expression in HPV16-positive samples. This discrepancy may be attributed to the relatively low levels of E6 expression observed in our cohort. Furthermore, it is possible that HPV suppresses mTOR expression as a strategy to evade immune surveillance, given that mTOR plays a central role in immune activation [76, 84].

Given the central role of this signaling cascade in breast carcinogenesis, inhibitors such as everolimus, in combination with exemestane, have been approved by the FDA for the treatment of metastatic, endocrine-resistant HR+/HER2− breast cancer with increased PI3K pathway activation [26]. Moreover, additional therapeutic strategies targeting other components of this pathway, including AKT inhibitors and mTORC1 inhibitors—the complex containing mTOR—are under development as potential treatments for breast cancer [26, 85–87]. Currently, these inhibitors are also being investigated for the treatment of HPV-positive malignancies, such as cervical cancer. A study by Lou et al. [88] (2026), using HPV-positive cervical cancer cell lines, demonstrated that inhibitors of the PI3K/AKT pathway, such as alpelisib and capivasertib, suppressed cellular proliferation, suggesting potential therapeutic benefit in advanced disease settings. Conversely, with regard to everolimus, a more favorable response has been observed in patients with HPV-negative head and neck cancer exhibiting higher mTOR expression [89]. Bossler et al. [84] demonstrated that HPV, particularly the E6 and E7 oncoproteins, can interact with and activate this signaling pathway in cervical cancer, representing a key mechanism in tumorigenesis. In this context, a comprehensive understanding of alterations in the effectors, activators, and inhibitors of this critical signaling cascade is essential to elucidate the role of HPV in breast cancer.

Cellular receptors such as EGFR activate PI3K, thereby positively regulating this signaling pathway. Upon activation, PI3K converts PIP2 into PIP3, leading to AKT phosphorylation and activation; in turn, active AKT promotes mTOR recruitment and activation [90]. Negative regulation of this pathway is mediated by the tumor suppressor PTEN, which dephosphorylates PIP3, thereby preventing AKT activation [91]. EGFR amplification is frequently observed in breast cancer, and increased EGFR copy number has been associated with poor prognosis, particularly in triple-negative breast cancer [92]. In cervical cancer, the E5 protein has been shown to promote cell proliferation by enhancing EGFR signaling [55]. In the present study, EGFR expression did not differ significantly between HPV16-positive and HPV16-negative breast cancer groups. However, correlation analysis revealed a positive association between EGFR and the viral oncogenes E5 and E7, supporting the hypothesis that these genes may contribute to immune evasion and tumor proliferation by modulating the host immune response—particularly through interactions with MHC I molecules, as previously reported in cervical cancer [93, 94]. Consistent with our findings, a recent study by São Marcos et al. [95] in HPV-positive lung cancer cells associated the expression of E5, E6, and E7 with enhanced immune evasion and tumor progression.

PTEN is a PIP3-specific phosphatase that dephosphorylates this molecule, converting it back to PIP2, thereby acting as a negative regulator of the PI3K/AKT/mTOR pathway [96]. PTEN prevents PIP3 from recruiting AKT, thereby inhibiting this signaling cascade, which, when hyperactivated, contributes to tumorigenesis; PTEN thus functions as a critical tumor suppressor. PTEN expression levels may be modulated by the E6 oncoprotein through degradation of discs large homolog 1 (hDlg), a member of the membrane-associated guanylate kinase (MAGUK) protein family known to regulate PTEN activity [69, 97]. The present study demonstrated a trend toward reduced PTEN expression in HPV16-positive samples, consistent with previous reports showing that HPV16 may interfere with PTEN levels, leading to its downregulation, embora que em ambos os casos não ocorrou diferença estatistica significativa [97, 98]. In this context, Naderi et al. [99] (2025), in a study of patients with HPV-positive breast cancer, reported decreased PTEN expression and associated this downregulation with a potential mechanism contributing to breast tumor progression. Collectively, these findings suggest that this may represent one of the mechanisms through which HPV contributes to dysregulated cell growth in breast cancer. Given that PTEN is a key regulator of this pathway, its reduced expression, together with increased PI3K expression, may promote enhanced cellular proliferation.

Overall, our PCA and gene expression heatmap summarize and highlight important interactions among the targets analyzed in this study. The PCA revealed the formation of three distinct groups: the first, comprising HPV16 oncogenes (E5, E6, and E7) together with the human genes PTEN and AKT (PC1); the second, consisting of E6, EGFR, and PI3K (PC2); and the third, strongly dominated by mTOR (PC3). This finding supports the association between HPV oncogenes and the PTEN and AKT genes, which may be related to modulation of immune responses and cell proliferation, particularly given the reduced PTEN expression observed in the HPV-positive group. Regarding PC2, the opposing signals between EGFR/PI3K and E6 suggest that this component separates samples with high expression of the receptor–PI3K axis from those with higher expression of the viral oncogene E6, possibly reflecting the dual role of this oncogene at different stages of pathway activation. Finally, mTOR formed an independent cluster from the other genes, which may help explain its downregulation despite PI3K upregulation in the presence of HPV. These findings suggest a potential compensatory mechanism or dysregulation of the PI3K/AKT/mTOR axis in HPV-infected breast cancer cells. This phenomenon, together with the higher expression of the E5 oncogene compared with E6 and E7 observed in our study, may indicate a potential role of HPV-mediated signaling modulation in immune evasion mechanisms in breast tumors, as previously observed in other HPV-positive cancers [100, 101].

Furthermore, the low expression of these genes may also suggest that HPV modulates signaling pathways independent of PI3K/AKT/mTOR, such as the MAPK and JAK/STAT pathways [102, 103]. Given the use of pathway inhibitors in the treatment of breast cancer, these findings highlight the need for further studies with larger sample sizes to better elucidate the role of HPV oncogenes in modulating these signaling networks, which may ultimately impact treatment response in HPV-positive breast cancer patients.