Neural crest cell signatures drive tumorigenesis in tuberous sclerosis complex and lymphangioleiomyomatosis
Uchenna J. Unachukwu, Enio B. Garcia, Nooralam Rai, Jeanine M. D’Armiento
Uchenna J. Unachukwu, Enio B. Garcia, Nooralam Rai, Jeanine M. D’Armiento
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Research Article Cell biology Clinical Research Oncology

Neural crest cell signatures drive tumorigenesis in tuberous sclerosis complex and lymphangioleiomyomatosis

Abstract

Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) lack well-defined cellular origins, limiting treatment options. In this report, scRNA-seq of Tsc2+/– mouse renal cystadenomas revealed an 80-fold increase in a tumor cell subpopulation with neural crest features, expressing known cranial neural crest genes as SRY box transcription factor 9 (Sox9), transcription factor activator protein (Tfap2a), and candidate neurocristopathy markers, osteopontin (Spp1), lipocalin-2 (Lcn2), clusterin (Clu), and cytokeratin 18 (Krt18). These signatures were validated in mouse tumors and LAM patient lesions and serum, identifying a tumor phenotype distinct from traditional VEGFD detection. Pathway analysis indicated activation of WNT/SHH signaling, nephric duct formation, and protumorigenic signals, with transcription factor 7 (Tcf7) and ephrin-A ligands as key upstream regulators. Spp1 KO in cranial neural crest cells (CNCCs) significantly reduced proliferation (28%–33%), migration (54%–76%), and invasion (29%–64%) without affecting viability, while Tsc2 KO increased viability 3- to 6-fold with minimal effect on chemotaxis. Elevated serum levels of SPP1 and KRT18 in 1 subset of patients with LAM, decreased LCN2 in nearly all cases, and distinct increases in VEGFD in a separate subset suggest complementary roles for these biomarkers. Overall, findings support a neurocristopathic model of tumor development in TSC and LAM and identify potential biomarkers and therapeutic targets beyond mTOR inhibition.

Authors

Uchenna J. Unachukwu, Enio B. Garcia, Nooralam Rai, Jeanine M. D’Armiento

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Figure 11

Tumorigenic effects of inhibiting Spp1 and Tsc2 expression in mouse CNCCs.

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Tumorigenic effects of inhibiting Spp1 and Tsc2 expression in mouse CNCC...
(A) CRISPR-based edits targeting exon 4 of Spp1 in 09-1 mouse CNCCs were confirmed by a PCR product size change at 935 bp. (B) qPCR validation of Spp1 suppression in CNCC clones (W5-A7, W5-B3, W5-B5) with KO-score/(insertion/deletion) (Indel%) of 94/(100%), 46/(48%), and 88/(88%), respectively. (C) Protein verification of Spp1-KO in CNCC clone W5-B5. (D–G) Spp1-KO did not affect clone viability, as measured by ATP luminescence (D) but significantly reduced CNCC proliferation (28%–33%) (E), invasion (29%–64%) (F), and migration (54%–76%) (G) compared with unedited W2-A8 CNCCs. Boyden chamber assays were initiated with 50,000 CNCCs per well and yielded similar results at lower cell densities (Supplemental Figure 13). (H) PCR confirmation results of CRISPR-mediated Tsc2 KO in exon 6 of 09-1 CNCCs in clones B1, B6, B7, B8, and C11 with KO-score/(Indel%) of 94/(94%), 92/(92%), 93/(93%), 93/(94%), and 92/(92%), respectively. (I and J) qPCR validation of Tsc2 downregulation (I), leading to a 25% proliferative increase in clones B1 and C11 (J). (K) ATP levels increased 3- to 6-fold across clones, indicating enhanced viability that may promote tumor survival and resistance. (L and M) Tsc2-KO minimally reduced migration (L) and invasion (M) in most clones. Results obtained using lower cell densities (10,000 cells/well) are described in Supplemental Figure 13. Each data point on the graphs represents the average across triplicate or quadruplicate wells per experiment, with at least 3 repeats. Statistics were performed using a 1-way ANOVA with Dunnett’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001.