Diagnostic Pathology: Open Access
Open Access

Lkb1 (Liver Kinase B1, encoded by Stk11) is a multifaceted serine/ threonine kinase recognized as a tumor suppressor, regulating cell metabolism, cell polarity, cell fate, and cell survival [1-6]. Previous studies revealed that global knockout of Stk11 in mice leads to embryonic lethality, indicating the pivotal role of Lkb1 in embryonic development [7]. Intriguingly, it is widely accepted that germline mutations in Stk11 are strongly associated with Peutz-Jeghers Syndrome (PJS) in humans, an inherited disease characterized by Gastrointestinal (GI) hamartomatous polyposis and increased risks for multiple types of cancers [8]. Subsequently, the exciting findings to manifest PJS-like intestinal polyposis in mouse models with heterozygous deletion of Stk11, have fueled the researchers worldwide to pursue the specific role of Lkb1 in the pathogenesis of PJS [9-11].

More and more conditional Lkb1 deletion mouse models have been developed to open new avenues for further investigation of the cell types and signaling pathways involved in polyp formation. Surprisingly, deletion of Lkb1 in intestinal epithelial cells results in increased susceptibility to colitis and suppression for microbial population, but shows no evidence of GI polyps in mice, even those 52 weeks of age [12,13]. These lines of evidence indicate the potential roles of nonepithelial Stk11/Lkb1 in the development of PJS-associated GI polyps. To this end, multiple conditional knockout mouse models of Lkb1 in mesenchymal (stromal) cells have been generated, including Tagln-Cre (Smooth Muscle Cell (SMC)-specific), Fsp1-Cre (Fibroblast -specific), Twist2-Cre and Gli1-Cre (mesenchymal progenitor cellspecific) Nkx3.2-Cre (pan-mesenchymal cell specific), which suggest that Lkb1 mutation in certain murine stromal cells could drive PJS-like GI polyposis N [14-16].

Our recent study further confirmed the critical role of mesenchymal Stk11/Lkb1 in the pathogenesis of gastrointestinal polyposis [17]. We generated tamoxifen-inducible Lkb1flox/+;Myh11-Cre/ERT2 (Lkb1 Het) and Lkb1flox/flox;Myh11-Cre/ERT2 (Lkb1 KO) mice. We found that heterozygous rather than homozygous Lkb1 deletion in murine mature SMCs is sufficient for the manifestation of PJS-like polyps, which is inconsistent with previous finding observed in mice with SMC-targeted inactivation of Stk11 by Tagln-Cre [14]. PJS-like polyps, characterized by an arborizing smooth muscle core, abundant ECM deposition and augmented immune cell infiltration, were observed in Lkb1 Het mice from 9 months post-tamoxifen treatment, in contrast to none developed in Lkb1 KO mice till their death. Furthermore, Lkb1flox/flox;Pdgfrα-Cre/ ERT2 mice, another mesen- chymal Stk11/Lkb1 deletion model, also simulated historically similar polyps to those in Lkb1 Het GI, as early as 2-3 months after tamoxifen treatment. Results supported the notion that Myh11+ or Pdgfrα+ mesenchymal cells may serve as an important cellular origin for PJS-like polyps.

To provide novel insights into the comprehensive cellular components and the underlying molecular mechanisms of the Lkb1- associated polyps, we performed a single-cell transcriptome atlas of Lkb1-associated polyps for the first time in Lkb1flox/+;Myh11-Cre/ERT2 mice. Clustering analysis revealed that there are polyposis-specific cell clusters and a higher portion of mesenchymal cells within Lkb1 Het duodenum polyps, compared with normal GI tissues. As the largest cell population in duodenum, the epithelial cells from Lkb1 Het polyp exhibited aberrant stem cell-like characteristics at an impaired differentiation state, along with an increment in expression of stem cell markers such as Cd44 as previously clarified but a decrement in mature enterocyte markers [15,18]. Of note, the up regulation of genes encoding secretory proteins of the gastric mucus barrier in those abnormal stem cell-like epithelial cells displayed the functional switch into a more secretary phenotype consistent with previous findings, which necessitates further research into the biological significance of Lkb1 in GI homeostasis.

Interestingly, coupled with the reported Spp1-Cd44 axis promoting tumor progression and metastases we found that intercellular communication networks (Spp1-Cd44 or Spp1-Itga8/Itgb1) among the epithelial, mesenchymal/stromal, and immune cells contribute to polyposis process [19,20]. Besides, special focus should be given to the abundant immune cell infiltration in Lkb1-related polyps in our study and other studies. Previous study demonstrated that Lkb1 deficiency in T cells is sufficient to promote the development of gastrointestinal polyps [13]. However, the underlying mechanism of deregulated inflammatory responses caused by Stk11/Lkb1 inactivation in stromal cells and immune cells is awaited to further identified.

In general, it is the first time to conduct a single-cell transcriptome atlas of Lkb1-associated polyps, trying to elucidate the pathological microenvironment changes, variations in cellular constitutions and functionalities and possible signaling pathways in cell-cell interactions. Key questions remain to be answered about how mesenchymal Lkb1 regulates epithelial cell fate/state in Lkb1-associated polyps. Further research is warranted in the aim of yielding clinical benefits for patients with PJS.

This work was supported by the National Natural Science Foundation of China (Grant Number: 82300489), the Shanghai Pujiang Program (Grant Number: 23PJD084), and the Nurture projects for basic research of Shanghai Chest Hospital (Grant Number: 2022YNJCQ03).

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