Pathogens, Vol. 12, Pages 33: Expression of HIF-1α and Genes Involved in Glucose Metabolism Is Increased in Cervical Cancer and HPV-16-Positive Cell Lines

1. IntroductionCervical cancer (CC) is the fourth leading cause of death in women worldwide, with approximately 604,127 new cases and 341,831 deaths annually [1]. Human papillomavirus 16 (HPV 16) is present in more than 50% of cases of CC [2]. The oncogenicity of HPV results mainly from the action of E6 and E7 oncoproteins. E6 promotes carcinogenesis by inducing the degradation of the tumor suppressor protein p53 and activating the PI3K/AKT/mTOR [3]. In contrast, E7 contributes to carcinogenesis by inducing the retinoblastoma tumor suppressor protein (pRb) degradation by releasing the cell cycle transcription factor E2F1 [4]. Several studies have revealed the association between HIF-1α overexpression and worse prognosis in patients with this type of cancer [5]. In different HPV-16-positive cancers, other active HIF1-regulated genes are overexpressed, which encode proteins that play an essential role in immortalization [6,7], cell proliferation, metastasis, and metabolic reprogramming [8].Metabolic reprogramming is an essential feature of cancer. Tumor cells reprogram their metabolism through a process known as the Warburg effect; this process is defined as the ability of cells to have high rates of glycolysis for the generation of ATP and precursors of different biomolecules independent of O2 [5,9]. The active HIF1 transcription factor is a master transcription factor of genes in response to hypoxia [10]. Active HIF1 is a heterodimer consisting of two subunits: HIF-α, which is induced by hypoxia and has three isoforms: HIF-1α, HIF-2α, and HIF-3α; and the HIF-1β subunit, which is constitutively expressed [10]. The transcription factors, such as the c-Myc-MAX heterodimer, the ISGF3 complex, composed of STAT1/STAT2/IRF9, STAT3, NF-κB, and even active HIF1 itself, bind to the promoter region of the HIF-1α gene to activate the initiation of its transcription [11]. Active HIF1 binds to the promoters of genes containing the 5′-RCGTG-3′ sequence, known as hypoxia response elements (HREs) [12]. Together with the coactivators CBP and p300 [13], they can regulate the expression of more than 70 genes, including some involved in glucose metabolism, such as HIF-1α, GLUT1, LDHA, CAIX, MCT4, and BSG (Basigin) [12,14]. HIF-1 upregulates the expression of GLUT1 and GLUT3 genes, both necessary for glucose uptake, and enhances lactate production by the enzyme lactate dehydrogenase A (LDHA), thereby decreasing intracellular pH. This mechanism regulates intracellular acidosis and is essential for maintaining homeostasis in the carbonic-anhydrase-IX (CAIX)-dependent mechanism [15]. Together with MCT4 and BSG, they facilitate the release of lactate and H+ to the extracellular milieu to neutralize intracellular acidosis in a HIF-1-dependent manner [16], which is associated with a hyperglycolytic and acid-fast phenotype in cancer [14,17,18] and the release of lactate into the extracellular milieu [19]. It has been shown in cancer in vitro that MCT4 is regulated by HIF1, unlike MCT1 and MCT2, because it has two HREs in its promoter [20].It has been reported that under hypoxia conditions in CC, E7 is associated with increased expression of HIF-1α via ROS, ERK1/2, and NF-κB [2,11]. In turn, E6 positively regulates HIF-1α, preventing its ubiquitination by VHL and subsequent degradation via proteasome [21]. Thus, E7 and E6 could indirectly promote increased glycolysis and resistance to intracellular pH changes through increased expression of HIF-1α and its target genes, such as GLUT1, LDHA, CAIX, MCT4, and BSG [3,9,22]. Moreover, the high glucose levels in blood have been associated with bad prognosis in CC, suggesting a potential relationship between CC and the metabolism of glucose [23]. However, the role of HPV 16 oncoproteins E6 and E7 in metabolic reprogramming is not entirely clear.

Therefore, in this work, we evaluated the expression of active HIF1 target genes: HIF-1α, GLUT1, LDHA, CAIX, MCT4, and BSG in HPV-16-positive CC cell lines and biopsies of CC patients using data from The Cancer Genome Atlas (TCGA). Likewise, an overall survival analysis was performed using the Kaplan–Meier Plotter database. Increased expression of HIF1A, SLC2A1, LDHA, SLC16A3, and BSG was observed in cell lines and biopsies of patients with CC and was associated with poor survival prognosis. This information will contribute to a better understanding of the mechanisms that favor metabolic reprogramming in HPV-16-positive CC.

4. DiscussionUnder hypoxia conditions in CC, it has been observed that HPV 16 oncoproteins E6 and E7 positively regulate HIF-1α. On the one hand, E7 promotes its gene expression, and on the other hand, E6 prevents its ubiquitination by VHL and its subsequent degradation via proteasome [21]. Several studies show that active HIF1 regulates gene expression in different hallmarks of cancer. The deregulated genes are grouped into tumor suppressor genes and oncogenes. In CC, deregulation of the expression of several genes is a mechanism that promotes tumor development and progression. These genes are known to code for proteins involved in processes such as metabolic reprogramming [9,22], angiogenesis [28], cell migration, invasion, and metastasis [29,30]. High expression of GLUT1, LDHA, and MCT4 proteins has been observed in biopsies from patients with invasive cervical cancer [31]. Additionally, LDHA inhibition has resulted in cell cycle inhibition and apoptosis in nasopharyngeal carcinoma [32]. It also suppresses cell migration, increases chemo- and radiosensitivity in cancer cells [33], induces cell cycle arrest in the G2/M phase, and activates the mitochondrial apoptosis pathway in CC cells [34]. On the other hand, there is evidence that HIF1A mRNA is overexpressed in CC [35] and laryngeal squamous cell carcinoma [36], while GLUT1 is overexpressed in CC [37] and colorectal cancer [38]. However, overexpression of LDHA, CAIX, MCT4, and BSG has only been reported in other types of cancer, but not in CC. LDHA overexpression has been reported in lung adenocarcinoma [39], CAIX in breast cancer [40] and oral squamous cell carcinoma [41], MCT4 in bladder [42] and breast cancer [43], and BSG in acute myeloid leukemia [44]. In this work, the expression of HIF1A, SLC2A1, LDHA, CA9, SLC16A3, and BSG was evaluated in tissue from patients with CC and normal tissue using the TCGA dataset and in HPV-negative (C-33A) and HPV-16-positive (SiHa and Ca Ski) cell lines.The expression of HIF1A, SLC2A1, LDHA, CA9, SLC16A3, and BSG was found to be increased in 306 samples from patients with CC compared with 13 samples of normal tissue. The increased expression of SLC2A1, LDHA, CA9, and SLC16A3 is statistically significant. However, the increase in HIF1A and BSG expression was not statistically significant. Increased expression was found for SLC2A1 [45], LDHA [46], CA9, SLC16A3, BSG [47] and HIF-1α [35,48]. Active HIF1 plays an important role in metabolic reprogramming in cancer by activating the transcription of genes encoding proteins involved in glucose metabolism, which promotes glucose uptake, conversion of pyruvate to lactate, pyruvate detour from mitochondria, and selective mitochondrial autophagy [49]. Active HIF1 regulates the expression of GLUT1, LDHA, CAIX [14], MCT4, and CAIX [12]. It can also bind to the promoter region of HIF-1α and promote its expression [11]. HIF-1α is the regulatory subunit in the formation of active HIF1, it is expressed under hypoxic conditions, and its expression is related to the Warburg effect in cancer [50]. These data suggest that the increased expression of HIF1A, SLC2A1, LDHA, CA9, SLC16A3, and BSG could be related to HIF1 activation triggered by increased HIF1A expression. These data were confirmed in the correlation analysis, where a positive correlation between the expression of HIF-1α and SLC2A1, LDHA, CA9, and SLC16A3 was observed. Interestingly, when HIFA1 expression is increased, SLC2A1, LDHA, CA9, and SLC16A3 expression is also increased. A negative correlation was observed between HIF1A expression and BSG expression, although the data were not statistically significant.On the other hand, in The Human Protein Atlas (https://www.proteinatlas.org/ accessed on 11 November 2022), gene expression data for HIF1A, SLC2A1, LDHA, SLC16A3, and BSG were found in 69 human cell lines. Interestingly, no data related to CA9 expression were found in SiHa, HeLa, and HaCaT cell lines (Figure S2). The data found show an increase in the expression of HIF1A, SLC2A1, and SLC16A3 transcripts, a slight increase in BSG expression, and a decrease in LDHA expression in the SiHa cell line, while in the HeLa cell line (HPV-18-positive), only increased expression of SLC16A3 was observed, compared to the immortalized human keratinocyte cell line (HaCaT). These data suggest that high-risk HPV could regulate the expression of HIF1A, SLC2A1, SLC16A3, and BSG genes in these BESEbases, which are required to carry out metabolic reprogramming. Importantly, no reports on the expression levels of these transcripts in the C33-A and Ca Ski cell lines were found on this platform.This work evaluated the expression of HIF1A, SLC2A1, LDHA, CA9, SLC16A3, and BSG transcripts in cervical cancer cell lines with and without HPV 16, compared to the HPV-negative C-33A tumor cell line, SiHa, with one to two integrated copies per cell of the HPV 16 genome, and Ca Ski with 600 integrated copies per cell of the HPV 16 genome. Increased expression of HIF1A, SLC2A1, LDHA, CA9, SLC16A3, and BSG transcripts was observed in the Ca Ski cell line compared to C-33A, with statistically significant differences in the expression of all genes evaluated, except CA9. In the SiHa cell line, there were only statistically significant differences in the expression of CA9 compared to C-33A. There is also an increase in the expression of HIF1A and SLC16A3 compared to C-33A; however, a slight decrease in LDHA and BSG was observed. When comparing the data obtained in this study with the data from the Human Protein Atlas, it is observed that there is a similar behavior in the expression of LDHA; however, it has been shown that in the SiHa cell line, E6 increases indirectly the LDHA expression via downregulation of miR-34a, which is a target of p53 [51]. On the other hand, the expression observed in the Ca Ski and C-33A cell lines was similar to that observed in the 306 CC samples and the 13 normal tissue samples reported in the TCGA dataset, in which the expression of the six genes evaluated was found to be increased with statistically significant differences in SLC2A1, LDHA, CA9, and SLC16A3 (Figure 1), as in primary advanced uterine cervical carcinoma [35], human-papilloma-virus-type-16-positive and negative cervical cancer, [37] and cervical cancer [42].These results support the theory that HPV 16 could be favoring the gene expression of HIFA1 through E7 and the formation of active HIF1 through E6 and that, in turn, active HIF1 may be inducing the expression of its target genes, such as HIF1A, SLC2A1, LDHA, CA9, SLC16A3, and BSG. This effect could be affected by viral load, E6, and E7 variants, or at the stage of tumor progression. Importantly, SiHa and Ca Ski cells, in addition to being HPV-16-positive, show two different stages of cancer progression, as they were derived from primary cervical carcinoma and metastatic tumor cells, respectively [52]. Ca Ski is a cervical cancer cell line established from cells from metastasis in the mesentery of the small intestine and containing the integrated HPV 16 genome of about 600 copies per cell and the E-G131/G350 variants of E6 and E7-Prototype [53]. On the other hand, the SiHa cell line was established from primary uterine squamous cell carcinoma tissue. It contained the integrated HPV 16 genome of one to two copies per cell and the E-G350/C442 variants of E6 and E7-C645 [54,55], given that the E6 variants of HPV 16 have an oncogenic potential difference and induce the differential expression of several genes [56].These particularities of the cell lines could explain why, although the two cell lines contain HPV 16, in Ca Ski, there is a high expression of HIF1A, SLC2A1, LDHA, SLC16A3, and BSG, whereas in SiHa, there is a higher expression of CA9.Changes in mRNA expression of various genes are often used to establish associations between gene transcription and disease stage. Previous studies have shown that high expression of LDHA is involved in cell proliferation and survival, migration, invasion, angiogenesis, and immune evasion in cancer, indicating that LDHA may be a potential prognostic marker and therapeutic target in cancer [7,57]. High SLC2A1 expression and HPV 16 have been reported to be independent prognostic factors in patients with CC [37]. On the other hand, increased expression of MCT1 and MCT4 is generally associated with poor prognosis. MCT4 is overexpressed in different types of cancer, such as breast, bladder, colorectal, and CC cancers. Moreover, high expression of MCT4 is closely associated with increased expression of CAIX and BSG [16,22].Regarding CAIX, its expression has been reported to regulate epithelial–mesenchymal transition and cell migration in CC [58]. In contrast, high expression of BSG has been correlated with radioresistance in the CC cell line SiHa [59]. Furthermore, it has been reported that shorter survival of patients in all types of breast cancer, especially in those with the triple-negative phenotype, is associated with high expression of HIF-1α [60], CA9 [61], and BSG [62]; LDHA in lung adenocarcinoma [39]; SLC16A3 in bladder cancer [42]; and SLC2A1 in colorectal cancer [38]. Likewise, in this study, the overall survival analysis with Kaplan–Meier curves shows that high expression of HIF1A, SLC2A1, LDHA, CA9, SLC16A3, and BSG is associated with worse survival in patients with CC (Figure 3) in which HPV 16 is the leading etiological agent. All these data support the theory that a higher expression of the transcripts evaluated here is associated with HPV 16 and a worse prognosis in CC.

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