Although it is considered extremely heterogeneous, AML has a unique origin from the transformation of HSCs into LSCs1,3. In recent years, it has been demonstrated that the BM niche plays an active role in myeloid leukaemic transformation and not only plays a passive role. Changes in this niche are believed to contribute to the initiation and progression of leukaemia in a model of malignant transformation guided by the haematopoietic niche7.
Several studies have described changes in hMSC-AML that culminate in haematopoietic insufficiency and the development of AML6,14,21,22. Therefore, this work focused on understanding the possible changes in signalling and/or biological processes in hMSC-AML that could be related to changes in HSCs and lead to leukaemic transformation and the onset of AML.
Initially, we determined that the hMSC-AML secretome is altered. After proteomic analysis of the supernatant from coculture assays, 15 differentially expressed proteins, including transcription factor, cytoplasmic, structural and secreted proteins, were identified. The presence of transcription factors among the identified proteins in the supernatant corroborates the literature evidence since it has already been established that, in addition to signalling through secreted proteins, hMSCs also signal from vesicles or exosomes. Therefore, hMSCs are capable of releasing these transcription factors into the extracellular environment23.
Given our focus on hMSC-AML-secreted proteins that could be involved in the transformation of HSCs into LSCs, we highlighted the increase in the SLPI protein in the supernatant of the AML condition compared to that in Healthy condition.
It is important highlighting that, all HSCs used in the AML condition were obtained from the same healthy donor, which were subjected to co-culture assays, under the influence of MSCs derived from different AML patients. Therefore, the only difference in the AML condition was in AML-MSC signaling, where we identified an increase in SLPI levels, potentially capable of modulating HSC gene expression.
A significant increase in the expression of SLPI was also observed in the bone marrow plasma from AML patients, corroborating the results found in the secretome. Furthermore, we detected an increase in SLPI expression in the transcriptome of isolated hMSC-AML cells compared to that in the transcriptome of hMSC-HD cells, suggesting that the increase in SLPI expression in the haematopoietic niche may be attributed to hMSC-AML signalling.
SLPI has already been described for its cancer-promoting ability, and its increased expression has been found in several tumours, including lung, pancreatic, gastric, breast and ovarian cancer, related to tumour progression13. Due to its presence in easily accessible fluids, such as saliva, blood and mucus, SLPI has been widely investigated as a potential diagnostic and prognostic biomarker for cancer23.
SLPI is expressed by a variety of cells, including granulocytes, monocytes/macrophages, and epithelial cells, and has always been considered a protease inhibitor protein. However, in recent years it has been described that its biological function is more complex. Research into this protein has expanded from basic biochemistry to studies of systemic diseases, and SLPI has been consistently reported to regulate gene expression. Thus, SLPI likely regulates the expression of many other genes, affects a variety of physiological and pathophysiological processes, and may act mainly as a transcriptional regulator24.
SLPI can be internalized into the cell and present cytoplasmic and nuclear localization15. In the nucleus, it can act as a transcription factor and bind to consensus sites in specific promoter regions in DNA, for example by binding to NF-kB sites in monocytes and inhibiting p65 binding25. Thus, it is possible that the increase in SLPI secretion by AML-MSCs could culminate in the regulation of genes in HSCs from healthy donors, and this would represent a new and intriguing avenue of investigation.
In cancer, SLPI has been shown to be associated with cell proliferation, apoptosis, invasion and metastasis24,26.
Klimenkova and colleagues observed that the absence of SLPI in HSCs derived from healthy donors is associated with reduced expression of several genes related to the regulation of myeloid differentiation, the cell cycle and proliferation. The authors showed that these cells exhibited cell cycle arrest and elevated apoptosis. To evaluate whether the increased expression of the secreted protein SLPI found in our study could be responsible for the alteration in gene expression described by Kimenkova, we performed transcriptomic analysis on HSCs obtained after coculture assays. Several SLPI targets presented increased expression in HSCs under AML conditions, corroborating the findings in the literature17.
Our results showed that important genes that regulate the cell cycle, such as MYC and the cyclins CCNA2, CCNE2, CCND2, and CDK1, were altered and that their expression was increased in HSCs from patients with AML. Cell cycle proteins are commonly altered in cancer and are often ideal targets for immunotherapy. Interestingly, MYC, which was also shown to be increased in our analysis, stimulates cell cycle progression and cell proliferation through the regulation of genes related to cell cycle control27. Increased expression has also been reported in more than 90% of a cohort of AML patients18. Overexpression of CCNE2, which was found in our analysis, has already been detected in cells from AML patients28. CDK1 promotes the G2/M and G1/S transitions, as well as G1 cell cycle progression, and is an indicator of malignancy in cancer20. Furthermore, CDKN2A is one of the most extensively studied tumour suppressor genes and plays a critical role in cell cycle progression, cellular senescence, and apoptosis29. Consequently, the suppression of CDKN2A induced by SLPI may facilitate accelerated HSC division.
Interestingly, CD133, also known as PROM1, is expressed at increased levels in acute myeloid leukaemia (AML) HSCs and has been documented as a marker of cancer stem cells (CSCs) in several human neoplasms, and its expression seems to predict unfavourable prognosis30, corroborating the hypothesis that increased SLPI may contribute to the leukaemogenesis process.
IGFBP-3 mediates antiproliferative and proapoptotic effects and acts as a tumour suppressor. It was shown that IGFBP-3 induces the growth arrest and apoptosis of human myeloid leukaemia cells31. We found that the expression of IGFBP-3 decreased in HSCs from patients with AML, demonstrating that SLPI may also play a role in cell proliferation induction and antiapoptotic processes.
In summary, our results showed that the secretome of hMSC-AML is altered, and the increased expression of the secreted SLPI protein may be associated with an altered gene expression profile in HSCs from healthy donors. These altered genes are associated with important biological processes in AML, such as the cell cycle, proliferation, and apoptosis, indicating that this protein could be important for leukaemic transformation in AML. However, whether these changes are related to the process of leukaemic transformation and/or progression of AML remains to be investigated.
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- Source: https://www.nature.com/articles/s41598-024-66400-7