Glycolysis-mTORC1 crosstalk drives rapid proliferation in patient-derived endometrial cancer spheroids with ALDH activity

doi:10.21203/rs.3.rs-3547380/v1

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Glycolysis-mTORC1 crosstalk drives rapid proliferation in patient-derived endometrial cancer spheroids with ALDH activity

https://doi.org/10.21203/rs.3.rs-3547380/v1

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Cancer stem cells are associated with aggressive phenotypes of malignant tumors. A prominent feature of uterine endometrial cancer is the activation of the PI3K-Akt-mTOR pathway. Herein, we present variations in sensitivities to a PI3K-Akt-mTORC1 inhibitor among in vitro endometrial cancer stem cell-enriched spheroid cells from clinical specimens. Intriguingly, the in vitro sensitivity mirrored effects observed in in vivo spheroid-derived xenograft tumor models. Our findings reveal a complementary suppressive effect on endometrial cancer spheroid cell growth when aldehyde dehydrogenase (ALDH) and PI3K-Akt inhibitors are combined. In the PI3K-Akt-mTORC1 signaling cascade, the influence of ALDH on mTORC1 is partially channeled through retinoic acid-induced lactate dehydrogenase A (LDHA) activation. LDHA inhibition was found to reduce endometrial cancer cell growth, paralleling the effects of mTORC1 inhibition. Building upon our prior findings highlighting ALDH-driven glycolysis through GLUT1 in uterine endometrial cancer spheroid cells, curbing mTORC1 bolstered glucose transport via GLUT1 activation. Notably, elevated LDHA expression correlated with adverse clinical survival and escalated tumor grade, especially in advanced stages. Collectively, our findings emphasize the pivotal role of ALDH-LDHA-mTORC1 cascade in the proliferation of endometrial cancer. Targeting the intricate interplay between mTORC1 and ALDH-influenced glycolysis could pave the way for novel strategies to combat this aggressive cancer.

Biological sciences/Cancer/Cancer stem cells

Biological sciences/Cancer/Gynaecological cancer/Endometrial cancer

Uterine endometrial cancer is a significant gynecological ailment. While early-stage and low-grade uterine endometrial cancers present relatively mild behaviors, standard chemotherapy regimens fail to adequately address high-grade or metastatic tumors (1). A common molecular characteristic of both type I and type II endometrial cancers is the dysregulation of the PI3K-Akt-mTORC1 signaling pathway (2). Positioned as a downstream target, mTORC1 is influenced by numerous oncogenic pathways in cancer, such as the PI3K/Akt and MAPK pathways (3). Furthermore, the mTORC1 complex is modulated by growth factors, amino acids, energy levels, and stress, and it plays a role in cell growth through protein, lipid, and nucleotide synthesis as well as autophagy (3). Similar to many cancers, most endometrial cancers exhibit genomic aberrations in the PI3K-Akt-mTORC1 signaling pathway, which often lead to mTORC1 hyperactivation (4). Thus, therapies targeting the PI3K-Akt-mTORC1 signaling could potentially enhance patient outcomes in endometrial cancer (3, 5). However, results from a recent phase I/II trial involving an mTOR inhibitor combined with an aromatase inhibitor, anastrozole, showed limited improvements in hormone receptor-positive recurrent or metastatic endometrial cancer patients, with an overall response rate of 24.5% (6). Another phase II clinical trial demonstrated that dual PI3K/mTOR inhibitors offered modest clinical benefits (overall response rate: 16%; duration of response: 4.2 months) with manageable side effects (7). Delving deeper into the mechanism of mTORC1 signaling and formulating methods to identify benefiting patients remains of paramount importance.

Cancer stem cells, identified as primary contributors to cancer origination, when eliminated, significantly retard cancer progression (8). Aldehyde dehydrogenase (ALDH) serves as a specific marker for many types of cancer stem cells (9). ALDH, pivotal in retinoid metabolism and toxic aldehyde removal, influences cancer progression, stemness, and chemotherapy resistance across various cancers. Notably, among the 19 ALDH isoforms, ALDH1A1, ALDH1A3, and ALDH3A1 are tied to cancer stem cells (10). We previously identified ALDH as a functional marker for both ovarian and uterine endometrial cancer stem cells (11, 12). In particular, we highlighted ALDH1A1 as the specific isoform marking uterine endometrial cancer stem cells and uncovering the role of the ALDH-GLUT cascade in paclitaxel resistance among these cells (12). However, comprehending the ALDH mechanism in endometrial cancer proliferation remains an open question.

Patient-derived tridimensional cells, encompassing spheroid and organoid cells from clinical specimens, retain several clinical traits. They are invaluable platforms for assessing drug sensitivity in numerous solid cancers, including ovarian and uterine endometrial cancers (13, 14). Our earlier research indicated that gynecological cancer patient-derived spheroid cells exhibit cellular diversity and potent tumorigenic capabilities in vivo, which are hallmark characteristics of cancer stem cells (11, 12, 15). Moreover, our spheroid cells have proven effective in drug sensitivity determination (16), echoing the utility of organoid cells that are universally recognized for drug sensitivity assays (17).

In this study, we delved into the sensitivity of inhibitors targeting the PI3K-Akt-mTORC1 signaling, employing uterine endometrial cancer patient-derived spheroid cells. We juxtaposed protein and genomic mutational profiles of these spheroid cells. A drug sensitivity test elucidated the interplay between ALDH and mTOR, uncovering the novel function of ALDH in cancer and its contribution to mTORC1 activation via LDHA. Furthermore, we identified the reciprocal relationship between glycolysis and mTORC1 in promoting the proliferation of endometrial cancer cells. These findings underscore that targeting ALDH-LDHA-mTORC1 signaling is a novel treatment strategy for aggressive uterine endometrial cancer.

Tumor-derived Spheroid Culture

Endometrial cancer spheroid cells isolated from clinical cancer specimens were cultured using ultra-low-attachment culture dishes (Corning, Corning, NY, USA). The culture medium was STEMPRO hESC SFM (Gibco, Grand Island, NY, USA) with a supplementation of 8 ng/ml basic fibroblast growth factor (Invitrogen, Carlsbad, CA, USA) and penicillin/streptomycin. Cells were maintained under specific conditions at 37°C with 5% CO₂ concentration (15). Spheroid cells were dissociated using Accumax (Innovative Cell Technologies, San Diego, CA, USA) for serial passaging. All the culture and handling protocols strictly adhered to the guidelines of the Ethics Committee of Niigata University and the National Cancer Center. Informed consent was duly acquired from all involved patients.

Animal Experiments

The drug efficacy experiments followed a randomized selection of mice into distinct groups. Accumax (Innovative Cell Technologies) was used to dissociate spheroid cells into single-cell structures. These cells were then suspended in a medium, comprising 50% Matrigel (BD Biosciences, San Jose, CA, USA; 3564234), before being subcutaneously injected using a 27-G needle into NOG (NOD/Shi-SCID-IL-2Rγnull) mice. These mice were sourced from the Central Institute for Experimental Animals, Kawasaki, Japan. The drug was administered every 2–3 days from 10–14 days after spheroid cell injection. The control group was exposed to dimethyl sulfoxide (DMSO) only. Mice were monitored every 3–4 days for a span of 4–6 weeks post-cell transplantation. The Animal Care and Use Committee of Niigata University approved all mouse procedures, ensuring they align with institutional policies.

Lentivirus-mediated Transduction

Both pCMV3-ALDH1A1 plasmid (HG11388-UT) and pCMV3 control vector were procured from Sino Biological (Beijing, China). The process of creating virus-containing supernatants and the subsequent viral infections were carried out as elaborated in a previously established protocol (11). Post-infection, cells underwent selection in a milieu containing 100 µg/ml hygromycin.

Western Blot Analyses

The cell lysis procedure was facilitated using radioimmunoprecipitation assay buffer. This buffer contained specific concentrations of several compounds, including 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1 mM ethylenediaminetetraacetic acid. To this mixture, protease and phosphatase inhibitors (Roche, Basel, Switzerland) were added. The resultant samples were then subjected to western blot analysis as detailed in a previous protocol (11), with the use of specific primary antibodies (Details provided in Supplementary Table 1).

Statistical Analyses

The statistical evaluation of both in vitro and in vivo spheroid cell experiments utilized Welch’s t-tests or Student’s t-tests, contingent on the outcomes of F tests. A threshold of p < 0.05 was set for significance. Clinical sample statistics were carried out using the EZR software (18). The Kaplan–Meier method was used for univariate survival analysis, whereas the significance of variances between groups was determined through log-rank tests.

Data Availability Statement

The microarray data analyzed in this study were obtained from the Gene Expression Omnibus database (accession number: GSE123530).

PI3K-Akt-mTOR signaling patterns in human uterine endometrial cancer spheroid cells

First, we evaluated the protein expression and gene mutation profile of PI3K-Akt-mTOR signaling to delineate the signaling status in endometrial cancer spheroid cells. Through western blot analysis of seven distinct endometrial cancer spheroid cells, we observed variations in the expression levels of signaling factors, including phospho-Akt, phospho-PTEN, and phospho-p70S6K. Intriguingly, their expression did not correlate with the ALDH1A1 expression within the spheroid cell types (Fig. 1A).

Subsequently, an immunohistochemical evaluation of spheroid-derived xenograft tumors was undertaken alongside the original clinical cancer tumor specimens. This highlighted that the expression patterns of phospho-Akt, phospho-p70S6K, and phospho-PTEN echoed the results observed in the aforementioned western blot analysis of spheroid cells. Notably, although a heterogeneous expression was discerned across tumors, a considerable number of cancer cells exhibited elevated phospho-Akt expression in tumors originating from or derivatives of EMN24 and EMN144 cells. Furthermore, the tumors from EMN24 cells displayed pronounced phospho-p70S6K expression, and the tumors from EMN108 cells showed evident phospho-PTEN expression (Supplementary Fig. S1A). In a broader context, mutation profiles across 22 endometrial cancer spheroid cells mirrored those witnessed in the original clinical tumor specimens (Fig. 1B) (12).

Among these PI3K-Akt-mTORC1-related signaling factors, a previous study showed that phospho-p70S6K is a predictive tool for the outcomes of patients with type II endometrial cancer when used as an immunohistochemistry (IHC)-based marker (2). This was particularly significant among markers related to the activation of the PI3K-Akt-mTORC1 signaling pathway (2). Furthermore, the phosphorylation observed at T389 showcased a connection with malignancy-related p70S6K activity (19). Merging this insight with the observed expression results of phospho-p70S6K in both spheroid cells and spheroid-derived xenograft tumors, we sought to confirm the expression of phospho-p70S6K in 35 clinically advanced endometrial cancer tissue specimens through immunohistochemical staining (Fig. 1C). Kaplan–Meier survival analyses showed that an elevated phospho-p70S6K expression may be correlated with overall survival (Fig. 1D, p < 0.05), not with progression-free survival (Supplementary Fig. S2A). The expression was not correlated with histological grade or clinical stage in these advanced-stage cases (Supplementary Fig. S2B and S2C, Fisher’s exact test). Though conclusions drawn from a limited case pool remain preliminary, our findings accentuate that phospho-p70S6K expression might indeed be intertwined with an adverse prognosis in advanced-stage uterine endometrial cancer.

PI3K Inhibitors curbing the proliferation of endometrial cancer spheroid cells

With the aforementioned results of phospho-p70S6K expression being related to the activation of the PI3K-Akt-mTORC1 signaling pathway (2), we then explored the effects of PI3K-Akt-mTORC1 signaling in endometrial cancer spheroid cells. Our in vitro cancer spheroid model has displayed superior efficacy in comparison to the cancer stem cell model (11, 12) and existing in vitro drug sensitivity evaluations (16). Riding on this fundamental advantage, we first assessed the sensitivity of endometrial cancer cells toward PI3K inhibitors. The particular focus was on Alpelisib, a prominent PI3K inhibitor deployed in clinical trials to treat breast and several other cancers (20, 21).

Upon assessment, we categorized the endometrial cancer cells into three distinct sensitivity groups with respect to Alpelisib:

High-sensitivity group: EMN18 and EMN21 cells.

Low-sensitivity group: EMN103 and EMN144 cells.

Intermediate-sensitivity group: Comprising the remaining cell types (Fig. 2A and 2B).

Our examination of other PI3K inhibitors, namely Taselisib and Copanlisib, showed sensitivity patterns similar to those observed for Alpelisib (Supplementary Fig. S3A and S3B). Alpelisib treatment enhanced caspase levels when the spheroid cellular growth was inhibited (Fig. 2B and 2C). To juxtapose in vivo sensitivity against its in vitro counterpart, we administered Alpelisib to NOG mice thrice weekly. The outcomes were illuminating: the xenograft tumors stemming from EMN18 and EMN21 spheroid cells (from the in vitro high-sensitivity group) exhibited a decline post-Alpelisib treatment (Fig. 2D). Moreover, tumors from EMN108 and EMN81 spheroid cells (intermediate-sensitive) also exhibited growth suppression (Supplementary Fig. S3C). Nonetheless, tumors originating from EMN144 cells (low-sensitivity group) remained unaffected by Alpelisib treatment (Supplementary Fig. S3D).

Collectively, these findings highlight that the Alpelisib sensitivity observed in our in vitro spheroid model mirrors the outcomes witnessed during in vivo applications.

Additive effects of PI3K and ALDH inhibitors on endometrial cancer spheroid cells' growth

Next, we elucidated the interactions between PI3K-Akt-mTORC1 signaling and cancer stemness using ALDH, which was found to be a functional marker of uterine endometrial cancer stem cells in our previous study (12). Intriguingly, a targeted sequencing analysis revealed identical mutational profiles between ALDH-high and ALDH-low spheroid cells (Fig. 3A). Moreover, despite the suppression of phospho-Akt and phospho-p70S6K expression post-Alpelisib treatment in sensitive spheroid cells (Fig. 3B), we observed no discernable differences in the in vitro sensitivity to Alpelisib between ALDH-high and ALDH-low cells (data not shown). This observation was consistent with the changes in ALDH activity or ALDH1A1 expression (Fig. 3B and Supplementary Fig. 4A). Further, Alpelisib treatment diminished phospho-p70S6K expression in both ALDH-high and ALDH-low cells (Supplementary Fig. S4B).

Given the above, we postulated that ALDH activity might be independent of PI3K sensitivity. To put this theory to the test, we co-treated spheroid cells with disulfiram (an ALDH inhibitor) and Alpelisib. Remarkably, this combination treatment led to a substantial inhibition of spheroid growth (Fig. 3C).

Validating these in vitro findings, we introduced this co-treatment to spheroid cell-transplanted mice. Tumors post-co-treatment were roughly half the size compared with those treated solely with Alpelisib. This was consistent for both high-sensitivity (EMN21, Fig. 3D) and intermediate-sensitivity (EMN24, Supplementary Fig. S4C) cell groups. Additionally, consistent with in vitro results, Alpelisib alone did not alter the ALDH activity in xenograft tumors (Supplementary Fig. S4D).

Conclusively, while ALDH activity appears to have no direct impact on PI3K inhibition, a combination of ALDH and PI3K inhibitors results in an additive inhibition of endometrial cancer progression.

Additive inhibition of endometrial cancer spheroid cells by combining Akt and ALDH inhibitors

Pivoting from our work with PI3K inhibitors, we turned our attention to the effects of Akt inhibitors on endometrial cancer spheroid cells. Notably, cells that exhibited low-to-intermediate sensitivity to PI3K inhibitors—specifically, the EMN81, EMN103, and EMN144 cell lines (Fig. 2A and Supplementary Fig. S3A and S3B)—responded to low-dose treatment with Akt inhibitors, Ipatasertib and Caplivasertib (Fig. 4A–4C). This sensitivity was marked by an uptick in activated caspases post-Ipatasertib treatment, implying cytotoxic effects (Fig. 4D).

However, it is worth noting that while EMN24 cell-derived xenograft tumors demonstrated resistance to Ipatasertib in vitro, they remained unresponsive to the drug even in vivo (Supplementary Fig. S5). Such findings underscore the potential of baseline Akt activity, especially linked to PTEN mutations, as a valuable predictor for Akt inhibitor sensitivity (Fig. 1A and 1B) (22).

While we observed a reduction in phospho-p70S6K expression after treatment with the Akt inhibitor, there was no noticeable shift in ALDH activity or expression (Fig. 4E). Such observations led us to hypothesize that ALDH activity remained unaffected by Akt inhibition. However, dual inhibition of both Akt and ALDH might collectively hinder endometrial cancer cell growth.

To test this, we investigated the combined impact of ALDH and Akt inhibitors, both in vitro and in vivo. The resulting data was as follows: combination treatment not only triggered apoptosis in endometrial cancer cells but also hindered their proliferation (Fig. 4F–4I). Mirroring our findings with PI3K inhibitors, this co-treatment approach further validated the additive repression of endometrial cancer progression, independent of any influence of ALDH activity on Akt inhibition.

mTOR inhibitor reduces the proliferation of endometrial cancer spheroid cells with ALDH activity

Drawing insights from our experiments with PI3K and Akt inhibitors, it became evident that PI3K/Akt activity was independent of ALDH activity. Our exploration then shifted toward understanding the influence of mTOR inhibition on endometrial cancer spheroid cell proliferation. Upon conducting in vitro sensitivity assays, we observed varying responses to mTOR inhibitors, everolimus, and Torin1. Notably, the range of sensitivities was more consistent than what was observed for PI3K and Akt inhibitors (Fig. 5A and Supplementary Fig. S6A). Following mTOR inhibitor administration, there was a decline in the levels of both phospho-p70S6K and phospho-4EBP1 across the spheroid cell population (Fig. 5B). Intriguingly, this decline in phospho-p70S6K was more accentuated in ALDH-high cells compared with ALDH-low cells (Fig. 5C and Supplementary Fig. S6B). Moreover, the ALDH-high cells exhibited heightened sensitivity to everolimus in comparison to their ALDH-low counterparts (Fig. 5D). It was discerned that the exogenous overexpression of ALDH1A1 further augmented this sensitivity to everolimus (Fig. 5E). Collectively, these findings bolster the notion that cells with higher ALDH activity inherently possess increased mTOR activation relative to cells with low ALDH activity. Alternatively, combination treatment with disulfiram and everolimus also hindered the spheroid cell proliferation (Fig. 5F). In conclusion, while ALDH activity appears to be linked to mTORC1 activity, a combination of ALDH and mTORC1 inhibitors additively impedes endometrial cancer progression.

Endometrial cancer spheroid cells exhibiting high ALDH activity show enhanced sensitivity to mTOR inhibitors

Based on the aforementioned results, we speculated that heightened ALDH activity might partially enhance mTOR sensitivity in endometrial cancer. To reveal the relationship between ALDH and mTOR, we examined changes in the expression of the PI3K-Akt-mTOR signaling pathway after manipulating ALDH activity. Disulfiram, which reduced ALDH activity, led to a decrease in phospho-p70S6K levels, whereas phospho-PI3K and phospho-Akt levels remained unchanged (Fig. 6A and Supplementary Fig. S7A). Additionally, gene set enrichment analysis (GSEA) revealed the ALDH-high cells preferentially expressed genes included in the gene set of hallmark of mTORC1 signaling (false discovery rate [FDR] q-value < 0.01, normalized enrichment score [NES] 1.59, p-value < 0.01) (23), genes upregulated after ectopically expressing eIF4E, and genes upregulated in control cells compared with eIF4GI-silenced cells (24, 25) (Fig. 6B and Supplementary Fig. S7B). Moreover, ALDH-high cells clearly expressed more phospho-p70S6K than ALDH-low cells (Fig. 6C). The exogenous overexpression of ALDH1A1 that led to ALDH activation (12) ultimately increased phospho-p70S6K levels (Fig. 6D). These results suggested that ALDH activity primarily affected mTORC1 activation rather than PI3K or Akt. To further confirm the relationship between ALDH and mTORC1 signaling, we determined whether mTOR activation could rescue the inhibitory effect of ALDH inhibitor on cancer cells. As expected, mTOR activator MHY1485 partially mitigated disulfiram-induced cytotoxicity in endometrial cancer spheroid cells (Fig. 6E, 6F, and Supplementary Fig. S7C–S7F). Moreover, western blot analysis indicated that MHY1485 could partially revert the disulfiram-mediated reduction in phospho-p70S6K expression (Fig. 6G and Supplementary Fig. S7G).

LDHA bridges ALDH activity and mTORC1 activation in endometrial cancer spheroid cells

To understand how ALDH influences mTOR signaling in endometrial cancer cells, we characterized its functional isoforms. Notably, of the 19 ALDH isoforms with analogous catalytic functions, ALDH1A1 predominantly dictates its activity (23). Our findings further cemented this observation, as endometrial cancer spheroid cells majorly expressed ALDH1A1, sidelining other ALDH isoforms (12). A pivotal function of ALDH1A1 includes the conversion of retinol to retinoic acids, driving cancer proliferation through multifaceted mechanisms (24, 25). Corroborating our premise that ALDH-mediated retinoic acid modulates endometrial cancer development in vitro, we discerned that additional retinoic acid rescued the cell mortality caused by disulfiram (Supplementary Fig. S8A–S8E). This mirrored the outcome when mTORC1 was activated with MHY1485 (Fig. 6E and 6F and Supplementary Fig. S7C–S7F). Experiments conducted on cells overexpressing ALDH1A1 further supported this observation (Supplementary Fig. S8F). Moreover, western blot outcomes indicated that retinoic acid could partially revert the disulfiram-mediated decrease in phopho-p70S6K expression (Supplementary Fig. S8G and S8H). This modulation paralleled the effects of the mTORC1 activator MHY1485 (Fig. 6G and Supplementary Fig. S7G).

To assess the influence of RA on mTOR activation, we combined GSEA results from microarray data and published RARA binding data from the ChIP-Atlas (http://chip-atlas.org/). Of 184 hallmark mTORC1 signaling genes (Fig. 6B) (26), 85 were core enrichment genes associated with ALDH-high endometrial cancer cells. Merging this with RARA-regulated genes from ChIP-Atlas, specifically those linked to malignancies, yielded eight candidate genes (Fig. 7A). Significantly, LDHA was predominantly expressed in ALDH-high cells (Fig. 7B). Delving deeper, ALDH1A1 overexpression augmented LDHA levels, whereas ALDH inhibition, using disulfiram, attenuated its expression (Fig. 7C, 7D and Supplementary Fig. S9A). Additionally, ALDH-high cells and exogenous ALDH1A1-overexpressing cells had higher LDHA activity than control bulk spheroid cells (Fig. 7E).

Upon investigating LDHA inhibition in cancer cells, we observed that using LDHA inhibitor AZ-33 considerably hampered endometrial cancer cell viability, especially in cells with ALDH activity (Fig. 7F). Interestingly, while the LDHA inhibitor diminished LDH activity and phospho-p70S6K expression, ALDH expression and its functional activity remained unaltered (Fig. 7G). Furthermore, treatment with AZ-33 led to a marked reduction in stemness indicators such as Nanog, Oct-4, and c-Myc (Fig. 7G). Additionally, activating mTORC1 with MHY1485 could partially counteract the inhibitory effects of AZ-33 on cancer cell proliferation, although MHY1485 in isolation did not make a significant difference (Fig. 7H and Supplementary Fig. S9B and S9C).

Taken together, our findings highlight that ALDH influences mTORC1 through LDHA, promoting the proliferation of endometrial cancer spheroid cells. Notably, while fluctuations in mTORC1 activation remained agnostic to LDHA levels, inhibiting mTOR with everolimus led to a surge in GLUT1. Conversely, stimulating mTOR with MHY1485 reduced GLUT1 levels (Fig. 7I and Supplementary Fig. S9D), influencing glucose transport (Fig. 7J and Supplementary Fig. S9E and S9F). These intricate interplays underscore a reciprocal relationship between glycolysis and mTORC1 in ALDH-active endometrial cancer progression (Supplementary Fig. S10).

LDHA expression is associated with adverse prognosis in endometrial cancer patients

Our study suggests that glycolysis and mTORC1 signaling play a pivotal role in regulating ALDH-high endometrial cancer cells. Seeking to delineate the distribution of ALDH and LDHA in a clinical framework, we performed immunostaining analyses on endometrial cancer samples. Intriguingly, ALDH-positive cells exhibited more pronounced LDHA expression than their ALDH-negative counterparts across both early and advanced cancer stages (Fig. 8A and Supplementary Fig. S11A).

To further elucidate the clinical implications of LDHA, serum LDH levels were quantified in 244 patients with endometrial cancer from our institution. Notably, LDH levels increased in patients with high-grade cancer relative to their low-grade counterparts across all stages, a trend particularly evident in stage I (Fig. 8B and 8C). Moreover, advanced-stage patients consistently exhibited elevated LDH titers in contrast to those in the early stages (Fig. 8D). This underscores a direct correlation between serum LDH levels, tumor grade, and clinical cancer progression.

Delving deeper into the clinical ramifications of LDHA expression, we utilized The Cancer Genome Atlas (TCGA) database (4) to investigate its correlation with the prognosis of patients with endometrial cancer. LDHA mRNA levels were discernibly elevated in high-grade tumors (Fig. 8E). Alarmingly, patients manifesting elevated LDHA expression experienced a significantly reduced overall survival (Fig. 8F, p = 0.02). Moreover, progression-free survival showed a concerning trend, with LDHA-high-expressing cases displaying a tendency toward shorter survival compared with their low-expression counterparts (Supplementary Fig. S11B, p = 0.09). A significant correlation was also evident between LDHA and RPS6KB1 expression in endometrial cancer tissue (Supplementary Fig. S8B, p = 0.01).

In summary, our findings highlight LDHA as a crucial biomarker, revealing its strong association with tumor grade and suggesting its potential role as an indicator of unfavorable prognosis in endometrial cancer.

The utilization of the patient-derived cell model offers promising avenues for understanding therapeutic responses to cytotoxic chemotherapeutic agents on an individual patient basis (27). Recent findings suggest organoid cells can effectively serve as models for gauging responses to therapies that target specific genetic mutations (28, 29). In alignment with these studies, our research highlights the correlation between in vitro and in vivo tests concerning the sensitivity to PI3K-Akt-mTOR signaling inhibitors in endometrial cancer patient-derived spheroid cells. Particularly, the response to PI3K and Akt inhibitors showed distinct differentiation based on genomic mutation profiles. These observations remark on the clinical potential of spheroid cells.

Endometrial, breast, and colon cancers often present activation in the mutant-dependent PI3K-Akt-mTOR signaling pathway (2, 4, 30). While PI3K-Akt-mTOR signaling inhibitors have theoretical potential to significantly suppress mutated cancer cells, challenges arise from negative feedback release and detrimental impacts on non-tumor cells. Furthermore, elements such as growth factors, energy, and stress play roles in mTOR activation (3). This drove our investigation into the non-genetic mechanisms underlying PI3K-Akt-mTOR pathway activation during endometrial cancer proliferation. The PI3K-Akt-mTOR pathway has been implicated in enhancing stemness, a trait linked to aggressive cancer manifestations (31). Numerous studies have pinpointed the role of PI3K signaling in cancer stemness, affecting markers such as Nanog, SOX2, and CD133 (32–34). For instance, mTORC1 activation has been noted to boost colon cancer stem cell proliferation (35). mTOR pathway is the downstream mediator of ALDH1A3 in gastric cancer (38). Our studies on uterine endometrial cancer emphasized the influence of both genomic and ALDH-mediated non-genomic aberrations on mTOR activation. Spheroid cells, enriched with cancer stem cells, proved instrumental in elucidating the nexus between cancer stemness and PI3K-Akt-mTORC1 signaling (36).

Lactate dehydrogenase (LDH), an intracellular enzyme, can manifest as either a homotetramer or heterotetramer, encompassing LDHA and LDHB subunits, giving rise to five isoforms. Each isoform is competent in transforming pyruvate to lactate (37). Echoing prior reports on cancers such as breast and pancreatic (38–40), our analysis attests to the association between LDHA expression and advanced-stage endometrial cancer prognosis. Furthermore, elevated LDHA expression and serum LDH levels were more prevalent in high-grade cancer patients. Past research has drawn parallels between elevated serum LDH levels and reduced survival rates in solid tumors such as melanoma and prostate carcinomas, highlighting its potential as a prognostic biomarker for metastatic carcinomas (41). Collectively, these insights underline the role of LDHA in exacerbating endometrial cancer progression and its clinical malignancy.

The interplay between glycolysis and cancer proliferation, especially processes such as epithelial-to-mesenchymal transition and cancer stemness, is the subject of extensive research (42). Our prior work revealed the centrality of the glucose transporter GLUT1 in dictating the stemness and chemoresistance traits of ALDH-active endometrial cancer stem cells (12). We later discovered the regulatory role of ALDH-mediated retinoic acid over glycolytic functional factor LDHA. LDHA is crucial for sustaining breast cancer stemness and triggering metastasis (39). Few studies have analyzed the role of LDHA in governing mTOR activation. In the context of pancreatic adenocarcinoma, LDHA-induced proliferation was found to be mediated by AMPK-mTOR signaling due to L-lactate (38). Other findings emphasize the role of LDHA in activating glycolysis, subsequently stimulating mTORC1 and thus fostering cellular proliferation. This phenomenon is particularly observed in K-Ras activated colon and pancreatic cancer cells (43). Furthermore, LDHA is required for the activation of mTOR in gastric cancer (44). Corroborating these findings, our research establishes that inhibiting LDHA impedes mTOR activation and curtails cancer cell proliferation, especially in ALDH-active endometrial cancer spheroids, regardless of K-Ras genomic aberrations (Fig. 7).

Moreover, our findings reveal the role of mTOR in modulating glycolysis. Curbing Akt-mTORC1 signaling diminishes GLUT1 expression, thereby stalling glycolysis and cancer cell survival in diseases such as leukemia (45, 46). The intricate dynamics between mTOR and glycolysis in cancer encompass both direct and indirect mechanisms. A standout feature of our study underscores the mutual regulatory relationship between glycolysis and mTOR activation. This feedback mechanism orchestrates the proliferation and survival of ALDH-active endometrial cancer stem cells (Fig. S10). Targeting this interplay could potentially enhance endometrial cancer treatment outcomes, even when mTORC1 activation is dictated by PI3K-Akt-mTOR genomic aberrations.

In summary, we disclosed the ALDH-LDHA-mTORC1 cascade to be a novel facet of the ALDH-related signaling that governs the proliferation of uterine endometrial cancer stem cells. Future studies should delve deeper into the cascade and interplay between glycolysis and mTORC1 to develop clinical treatment strategies to tackle the outcome of aggressive uterine endometrial cancer.

Additional materials and methods can be retrieved in Supplemental Experimental Procedures.

Author Contributions

Conception and design: H.U., T. I.

Development of methodology: H.U., T.I.

Acquisition of data: H.U., T.I., Y.M.

Analysis and interpretation of data: H.U., T.I., Y.M., K.Yamawaki

Writing, review, and/or revision of the manuscript: H.U., T.I., Y. M., K.Yamawaki, K. Yoshihara, K. O., and T.E.

Study supervision: T. E., K.Y.

Acknowledgments

We thank Ryo Tamura, Kazuaki Suda, Nozomi Yachida, Manako Yamaguchi, Kentaro Sugino, Tomoyuki Sekizuka, Kyota Saito, Anna Ishida (Department of Obstetrics and Gynecology, Niigata University Medical School) for scientific advice and technical assistance. This research was supported by Grants-in-Aid for Scientific Research (C) (Grant No. 18K09250, 21K09490) to T.I. from the Japan Society for the Promotion of Science. This study was partially supported by the National Cancer Center Research and Development Fund (29-A-2).

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There is NO conflict of interest to disclose.

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Glycolysis-mTORC1 crosstalk drives rapid proliferation in patient-derived endometrial cancer spheroids with ALDH activity

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Abstract

Figures

Introduction

Materials and Methods

Tumor-derived Spheroid Culture

Animal Experiments

Lentivirus-mediated Transduction

Western Blot Analyses

Statistical Analyses

Data Availability Statement

Results

PI3K-Akt-mTOR signaling patterns in human uterine endometrial cancer spheroid cells

PI3K Inhibitors curbing the proliferation of endometrial cancer spheroid cells

Additive effects of PI3K and ALDH inhibitors on endometrial cancer spheroid cells' growth

Additive inhibition of endometrial cancer spheroid cells by combining Akt and ALDH inhibitors

mTOR inhibitor reduces the proliferation of endometrial cancer spheroid cells with ALDH activity

Endometrial cancer spheroid cells exhibiting high ALDH activity show enhanced sensitivity to mTOR inhibitors

LDHA bridges ALDH activity and mTORC1 activation in endometrial cancer spheroid cells

LDHA expression is associated with adverse prognosis in endometrial cancer patients

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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