Abstract
Graphical abstract
Abstract
Lymphangioleiomyomatosis (LAM) is a cystic lung disease found almost exclusively in genetic females and caused by small clusters of smooth muscle cell tumors containing mutations in one of the two tuberous sclerosis genes (TSC1 or TSC2). Significant advances over the past 2–3 decades have allowed researchers and clinicians to more clearly understand the pathophysiology of LAM, and therefore better diagnose and treat patients with this disease. Despite substantial progress, only one proven treatment for LAM is used in practice: mechanistic target of rapamycin complex 1 (mTORC1) inhibition with medications such as sirolimus. While mTORC1 inhibition effectively slows LAM progression in many patients, it is not curative, is not effective in all patients, and can be associated with significant side effects. Furthermore, the presence of established and accurate biomarkers to follow LAM progression is limited. That said, discovering additional diagnostic and treatment options for LAM is paramount. This review will describe recent advances in LAM research, centering on the origin and nature of the LAM cell, the role of estrogen in LAM progression, the significance of melanocytic marker expression in LAM cells, and the potential roles of the microenvironment in promoting LAM tumor growth. By appreciating these processes in more detail, researchers and caregivers may be afforded novel approaches to aid in the treatment of patients with LAM.
Introduction
Lymphangioleiomyomatosis (LAM) is a rare chronic disease whereby the lungs of affected individuals progressively develop large cysts that replace normal alveolar airspace, resulting in a steady loss of lung function that, in some circumstances, leads to lung transplantation (Ferrans et al. 2000, Kelly & Moss 2001). Interestingly, the presence of these cysts appears to be due to a myriad of microscopic smooth muscle cell-like tumors that spread throughout the lungs of LAM patients; thus, LAM is considered a slow-growing multifocal malignancy (Zhe & Schuger 2004, McCormack et al. 2012).
There are four tenets of LAM that afford us insight into its pathogenesis and potential therapeutic targets. First, LAM has an extreme female sexual dimorphism, as nearly every significant case of LAM occurs in genetic females (Aubry et al. 2000, Miyake et al. 2005, Taveira-DaSilva et al. 2010, Zhang et al. 2022). Related to this sexual dimorphism, clinical evidence indicates that LAM progression may be estrogen sensitive, as LAM first becomes apparent after puberty, worsens with estrogen use or pregnancy (Shen et al. 1987, Brunelli et al. 1996, Johnson & Tattersfield 1999), and often stabilizes after menopause (Johnson & Tattersfield 1999, Lu et al. 2017). In addition, LAM tumor cells express estrogen and progesterone receptors (Berger et al. 1990, Kinoshita et al. 1995), and animal, as well as in vitro, models of LAM demonstrate various levels of estrogen sensitivity (Yu et al. 2004, 2009, Prizant et al. 2013, Sun et al. 2014a, Prizant & Hammes 2016, Prizant et al. 2016, Obraztsova et al. 2020). Second, LAM tumor cells contain mutations in one of two tuberous sclerosis genes (TSC1 or TSC2), leading to constitutive activation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway (Smolarek et al. 1998, Carsillo et al. 2000) that drives tumor growth (Goncharova et al. 2002). Statistically, the majority of individuals with LAM have germ cell mutations in TSC1 or TSC2 as part of the multisystem disease tuberous sclerosis complex (Taveira-DaSilva et al. 2010, Hammes and Krymskaya 2013), with only 15–20% having sporadic (nongermline) mutations in TSC genes. However, these numbers are reversed with regard to severity of disease – approximately 80–85% of patients with clinically significant LAM have sporadic (nongermline) mutations (Ryu et al. 2006, Tobino et al. 2015). The discovery of mutations in the tuberous sclerosis genes has led to the use of mTORC1 inhibitors such as sirolimus in LAM treatment (McCormack et al. 2011, McCarthy et al. 2021). While mTORC1 inhibitors often slow LAM progression, they do not reverse the cystic change or eliminate the LAM tumors, are ineffective in some patients, and can have side effects that preclude their use; thus, developing alternative LAM treatments is critical. Third, LAM cells express proteins normally found in melanocytes, such as premelanosome protein (PMEL) and glycoprotein NMB (GPNMB) (Kuhnen et al. 2001, Zhe & Schuger 2004, Klarquist et al. 2009, Prizant et al. 2016). These melanocytic markers have been used in tissue samples to differentiate LAM from other cystic lung diseases; however, why these proteins are expressed and how they function in LAM tumors is not understood. Finally, LAM appears to be metastatic in nature, as LAM cells are found in blood and lymph of LAM patients, and patients who have undergone lung transplantation often have recurrence of LAM in transplanted lungs (Bittmann et al. 2003, Karbowniczek et al. 2003).
Here we describe some important unanswered questions facing LAM research and how addressing these questions may guide LAM treatment. We focus on understanding the origin of the LAM cell and whether this knowledge can help explain the sexual dimorphism and estrogen sensitivity of LAM. We also examine the melanocytic markers in LAM cells and whether they can be used as diagnostic tools or therapeutic targets in LAM treatment. Finally, we discuss the tumor microenvironment – specifically, how immune cells within the tumor niche regulate tumor growth and therefore could serve as targets to treat LAM.
Origin and nature of LAM cells
To appreciate how TSC2-null smooth muscle-like LAM cells promote disease, it is first important to delineate their origin. Some hypothesized sources of LAM tumor cells include the kidney, neural crest, mesenchyme, and uterus. With regard to the kidney, LAM tumor cells share phenotypic, genetic, and transcriptomic features with renal angiomyolipomas (AMLs) found in the same patients, suggesting linkage (Carsillo et al. 2000, Guo et al. 2020); however, AMLs are present in similar proportions in males and females, as opposed to the profound sexual dimorphism of LAM. As for a potential neural crest origin, LAM cells express markers seen in cells of neural crest origin (Delaney et al. 2014); however, again, a neural crest origin does not explain the sexual dimorphism and estrogen sensitivity of LAM.
Single-cell RNA sequencing of human lungs has helped narrow the discussion regarding the LAM cell origin to lung and/or uterine mesenchyme. In one human single-cell RNA sequencing analysis, investigators found a distinct population of mesenchymal cells in LAM patients but not healthy controls that expressed myosin heavy chain 10 (a smooth muscle marker), ERα and ERα transcriptional target genes, and VEGFD (a known biomarker for LAM) (Obraztsova et al. 2020). Based on these data, investigators created a mouse model whereby TSC2 was knocked out primarily in the lung mesenchyme. These mice developed cystic lung disease that shared LAM characteristics of pulmonary decline and increased mTORC1-mediated signaling. Female mice had more progressive loss of lung function than males, and demonstrated differential changes in mesenchymal gene expression (Obraztsova et al. 2020), suggesting a possible sexual dimorphism. However, progressive increase in alveolar size was the same in males and females. Furthermore, direct evidence of estrogen-sensitive cystic lung disease and TSC2-null tumor growth in female lungs, as well as expression of melanocytic markers, has yet to be demonstrated in this mouse model.
Another single-cell RNA sequencing study focused on the ‘LAMCORE’ cells present in LAM vs control lungs that lacked TSC2 mRNA expression while also expressing 17 genes unique to LAM cells (Guo et al. 2020). Transcriptomic analysis of the LAMCORE cells demonstrated a uterine profile, and, unlike the abovementioned mesenchymal cell population, showed high expression of known LAM markers PMEL, MLANA, GPNMB, ESR1, and HOXgenes. These human data confirmed a previous mouse model whereby TSC2 was specifically knocked out in the mouse uterus, leading to LAM-like TSC2-null myometrial tumors with elevated mTORC1 signaling and expression of ESR1, PMEL, MLANA, GPNMB, and other melanocytic markers (Prizant et al. 2013, Prizant et al. 2016). TSC2-null myometrial tumors were suppressed in the absence of estradiol, and 50% of uterine-specific TSC2-null mice developed myometrial metastases to the lungs. Of note, mice with metastatic lung disease did not develop cystic lung disease, indicating that, while this model may be useful to study LAM tumor cells, it is not a model for cystic LAM. Nonetheless, these human and mouse data suggest that the LAM lung cells may be metastatic from the uterine myometrium or at least share a common developmental pathway, a concept supported by the presence of myometrial LAM clusters in 90% of uteri removed from LAM patients (Hayashi et al. 2011).
In sum, these studies suggest that both cells of uterine and lung mesenchyme origin may be involved in the development of LAM – perhaps the former comprise the core smooth muscle cell-like LAM tumor cells, while the latter are responsible for the cystic lung disease. More studies that include use of a newly developed LAM Cell Atlas (Du et al. 2023) will help clarify these questions.
Estrogen and LAM
As mentioned in the ‘Introduction’ section, clinical evidence suggests that LAM progression may be estrogen sensitive, although the significance of this estrogen sensitivity remains controversial. To address this debate, investigators have employed a variety of in vivo and in vitro systems to examine estrogen’s role in modulating LAM-like tumor cell actions. For instance, estradiol-enhanced lung colonization of TSC2-null Eker rat leiomyoma-derived (ELT3) tumor cells when mice were burdened with flank tumors or injected with tumor cells via tail vein (Yu et al. 2009). Further, aged mesenchyme-specific TSC2-null breeding female mice demonstrated exacerbated alveolar expansion and respiratory decline when compared with nonbreeding females, suggesting that estrogen surges during pregnancy may worsen cystic disease in mice (Obraztsova et al. 2020). Finally, estrogen ablation in uterine-specific TSC2-null knockout mice regressed uterine growth and reduced mTORC1 signaling (Prizant et al. 2013, 2016). Bulk mRNA sequencing in TSC2-null uteri from untreated and estradiol-exposed mice revealed significant estradiol-regulated transcriptome-level changes, including upregulation of melanocytic markers such as GPNMB as well as markers of immune infiltration, the latter indicating that the tumor immune microenvironment may play a role in the estrogen sensitivity of LAM-like tumor growth.
Established in vitro models – namely, cells derived from AML lesions of a LAM patient (621.101 cell line) and rat ELT3 cells – have also been used to assess the effects of estradiol on TSC2-null tumor cells (Sun et al. 2014a,b, Li et al. 2016, Prizant et al. 2016). Both cell lines demonstrate variable estradiol-mediated increases in transcription-dependent and transcription-independent signaling, estradiol-mediated changes in intracellular metabolism, and estrogen-enhanced proliferation, migration, and invasion. Worth mentioning, while statistically significant, some of these estradiol-mediated actions remain modest relative to in vivo mouse studies, suggesting potential limitations in using current TSC2-null cell lines when studying estrogen sensitivity.
In humans, randomized, controlled intervention studies lowering estradiol in LAM patients are limited to one: The Trial of Aromatase Inhibition in Lymphangioleiomyomatosis (TRAIL) (Lu et al. 2017). This study, designed to assess the effectiveness of aromatase inhibition in postmenopausal LAM patients, did not detect significant effects of aromatase blockade; however, fewer than 20 individuals enrolled in TRAIL, and most were already on sirolimus. Furthermore, lung function decline was minimal in all women, most likely since they were already postmenopausal with low estradiol exposure. That said, the breadth of clinical observations and preclinical evidence supporting estrogen as a driver of LAM progression warrants revisiting a trial of estrogen ablation in premenopausal women.
Melanocytic markers
Another common feature of LAM is the expression of melanocytic markers in TSC-null LAM tumor cells; therefore, examination of the role of these markers in LAM is warranted. Melanocytic markers are expressed primarily in skin melanocytes and upregulated in melanoma. In fact, they are used as histological and serum markers for melanoma (Hoashi et al. 2010), and melanocytic markers such as PMEL have been used to identify LAM cells in the lungs and other tissues (Kuhnen et al. 2001). However, the question as to why melanocytic markers are upregulated and what their function is in LAM is unclear.
Microphthalmia-associated transcription factor (MITF) is a master regulator of multiple melanocytic markers. In normal melanogenesis, MITF activates pigmentation genes such as PMEL and DCT. In melanoma, MITF has been shown to activate antiapoptotic BCL proteins, promote proliferation, and mediate endolysosomal biogenesis and WNT signaling (Ploper et al. 2015). In TSC-deficient cells, transcription factor EB (TFEB), a member of the family of MITF transcription factor family with similar abilities to upregulate other melanocytic markers, is hypophosphorylated and nuclear, leading to lysosomal biogenesis and cell proliferation (Alesi et al. 2021). In human TSC-associated renal adenocarcinomas and pulmonary LAM nodules, TFEB is similarly nuclear, perhaps partially explaining the presence of melanocytic markers in LAM cells. Importantly, GPNMB is a known target of TFE3 and TFEB (Baba et al. 2019) (Fig. 1).
Melanocytic markers in the LAM tumor microenvironment. Highlighting the LAM cell and melanocytic marker expression and interactions, (A) MITF is a master regulator of melanocytic marker expression and acts as a transcription factor to transcribe other melanocytic marker genes such as GPNMB. TFEB is increased in TSC-null LAM cells and can similarly drive transcription of melanocytic markers. Here GPNMB is shown to be expressed both intracellularly and on the cell membrane, and its ectodomain can be cleaved off the cell surface. (B) GPNMB in its soluble or membrane-bound form can interact with other LAM cells to potentially increase metastasis. (C) Soluble GPNMB or GPNMB expressed on immunosuppressive monocytes can interact with syndecan-4 to suppress T cell function.
Citation: Endocrine-Related Cancer 30, 9; 10.1530/ERC-23-0102
In the uterine-specific TSC2-null mouse model, melanocytic mRNAs encoding Pmel, Mlana, Gpnmb, Mitf, and Dct are upregulated, and expression increases with age and uterine weight relative to wild-type mice (Prizant et al. 2016). Expression was nearly abrogated with oophorectomy, letrozole, or rapamycin treatment, indicating that melanocytic marker expression in this model was exquisitely sensitive to estradiol and mTORC1 signaling (Prizant et al. 2016). Notably, GPNMB was found to be expressed in LAM lesions from both the lungs and uteri of LAM patients (Prizant et al. 2016). In fact, single-cell RNA sequencing data of human LAM showed that GPNMB mRNA is one of the most highly expressed mRNAs in LAMCORE cells (Guo et al. 2020).
Regarding GPNMB’s function in cancer, evidence suggests that GPNMB promotes metastasis in various cancers such as prostate, lung, breast, and pancreatic (Torres et al. 2015, Oyewumi et al. 2016, Rose et al. 2017). Furthermore, GPNMB expression positively correlated with worse prognosis and metastasis in triple-negative breast cancer (Huang et al. 2021). Interestingly, GPNMB is a differentially expressed antigen present primarily intracellularly in benign melanocytes but both intracellularly and on the cell surface of tumor cells such as melanoma, triple-negative breast cancer, and glioblastoma (Rose et al. 2010, Chung et al. 2014, Taya & Hammes 2018). In addition, the GPNMB ectodomain has been shown to be released from the cell surface of tumor cells. These observations suggest that GPNMB could be a mediator of LAM cell metastasis, a potential cell surface target for LAM treatment, and a possible biomarker for LAM tumor burden.
As mentioned, differential melanocytic marker expression in LAM cells underscores the potential use of immunotherapy in treating LAM. Cancers often evade the immune system by expressing self-antigens. However, sometimes tumor cells differentially express proteins such as melanocytic markers that can serve as targets for potential treatment. For example, PMEL was used as a target in an adoptive T cell transfer experiment to decrease LAM-like tumor growth in vitro and in mice (Han et al. 2020). Cell surface GPNMB and the soluble GPNMB ectodomain also interact with syndecan-4 on T cells to suppress T cell function and proliferation (Chung et al. 2007, Ramani et al. 2018) (Fig. 1). Finally, blocking GPNMB expressed on the cell surface of immunosuppressive monocytes with an anti-GPNMB antibody rendered T cells from cancer patients functional again (Kobayashi et al. 2019).
The immune microenvironment and LAM
The potential interactions between melanocytic markers and the tumor immune microenvironment highlight the importance of investigating the LAM nodule microenvironment and its unique tumor-supporting immune landscape. In fact, researchers have begun to make a case for potentially employing immune targeting therapies to attenuate LAM tumor growth, with hopes of perhaps eliminating the TSC2-null LAM cells entirely (Fig. 2).
LAM tumor immune microenvironment consists of potential therapeutic targets. Comprehensive analysis of LAM lungs isolated from patients using single-cell RNA sequencing demonstrates an immunologically diverse microenvironment consisting of LAM-associated fibroblasts (LAFs), monocytes, macrophages (including prealveolar, alveolar, and interstitial macrophages), dendritic cells, B cells (including plasma cells), CD4+ T cells, CD8+ T cells, T regulatory cells (Tregs), natural killer (NK) cells, mast cells, and neutrophils. Use of human LAM samples and various preclinical models have identified potential immune cell-related targets to either combat the immunosuppression or direct promotion of tumor growth mediated by the noted immune cells.
Citation: Endocrine-Related Cancer 30, 9; 10.1530/ERC-23-0102
Much of the work related to the LAM tumor immune microenvironment focuses on adaptive immune cells such as cytotoxic T cells. Many malignancies escape immune-mediated destruction by reducing tumor cell immunogenicity and suppressing antitumor T cells (Blankenstein et al. 2012). While LAM is considered to have limited antigenicity, some studies report elevated expression of CD3e and PD-1, markers indicative of T cell infiltration and exhaustion, respectively (Liu et al. 2018, Maisel et al. 2018, Han et al. 2020). The aforementioned adoptive T cell transfer study demonstrated heightened expression of PD-1 in PMEL-reactive CD8+ cytotoxic T cells (Han et al. 2020), and combatting T cell exhaustion in vivo with anti-PD-1 treatments caused tumor regression. Other studies similarly demonstrated reduction in tumor growth in mouse models for LAM with anti-PD-1 treatment alone or coupled with anti-CTLA4 (Liu et al. 2018, Maisel et al. 2018), together suggesting that checkpoint inhibitors may play a future role in LAM treatment. Chimeric antigen receptor (CAR) T cell therapy has also been proposed as an anti-LAM treatment strategy; one investigation described enhanced tumor-free survival in mice receiving adoptive transfer of CAR T cells engineered to bind ganglioside D3, a protein highly expressed on TSC2-inactive tumor cells (Thomas et al. 2021). Finally, an examination of AML- and LAM-associated tumors using single-cell transcriptomic modalities demonstrated that TSC-null tumors consist of inflammatory state (IS) and stem-like state (SLS) tumor cells (Tang et al. 2022). IS tumor cells were associated with low expression of midkine (MDK), resulting in increased immunogenicity, elevated expression of type 1 inflammatory cytokines, and increased clonal expansion of cytotoxic T cells. In contrast, SLS tumor cells were associated with high expression of MDK, which increased expression of APOE – a protein associated with enhancing the macrophage immunosuppressive (M2) program (Baitsch et al. 2011, Kemp et al. 2021) – and suppressed cytotoxic T cell clonal expansion. Of note, checkpoint inhibitors and CAR T cell therapies come with their own set of risks and may not be immediately amenable to LAM treatment; however, the concepts gained from understanding how LAM tumors evade adaptive immunity will likely result in safer, targeted approaches in the future.
In addition to suppressed adaptive immunity playing a role in LAM progression, innate immunity may also be important. For example, LAM tumor nodules sampled from the lungs of LAM patients demonstrated high expression of UL-16 binding proteins 2 (ULBP2) and 3 (ULBP3), which are ligands for the natural killer (NK) cell–activating receptor NKG2D. In addition, soluble forms of ULBP2 and ULBP3 were detected in the serum of LAM patients, and levels positively correlated with respiratory function decline in LAM patients. Finally, LAM patients in this study had fewer circulating NKG2D+ NK cells. These observations suggest that LAM cell shedding of NKG2D ligands may mediate tumor cell evasion of NK cell immune surveillance (Osterburg et al. 2016).
Mast cells, innate immune cells of myeloid lineage, have also been implicated in LAM progression. Using 3D spheroid cultures and samples from LAM patients, a study demonstrated that LAM tumor cells activated secretion of mast cell-recruiting chemokines, causing mast cell accumulation into the spheroids. In fact, mast cell presence in LAM nodules was positively associated with lung function decline. Finally, in vitro, mast cells within spheroids released the serine protease tryptase, and treatment with mast cell or tryptase inhibitors attenuated mast cell-mediated spheroid growth (Babaei-Jadidi et al. 2021).
Another innate immune cell of myeloid origin, the neutrophil, also may be mediating LAM progression. Immunophenotyping of wild-type and uterine-specific TSC2-null mice revealed tumor burden–induced neutrophil accumulation in the blood, bone marrow, lungs, spleen, and uteri, resulting in a tumor immune microenvironment that is myeloid cell rich with a neutrophil predominance, both indicators of immunologically cold tumors (Taya et al. 2020, Minor et al. 2023). These data, coupled with evidence of enhanced neutrophil expansion from the bone marrow progenitors isolated from these tumor-burdened mice, suggest that TSC2-null tumor growth induces granulopoiesis (Minor et al. 2023). In addition, tumor-associated neutrophils in uterine-specific TSC2-null animals were in fact important positive regulators of myometrial tumor growth, as treatment with a Gr-1+ cell-depleting antibody to reduce myeloid cell expansion, a CXCR2 antagonist to inhibit neutrophil recruitment, and a neutrophil elastase (NE) inhibitor all significantly reduced TSC2-null myometrial growth. Furthermore, NE enhanced TSC2-null cell proliferation, migration, and invasion in vitro. Together, these data indicate that neutrophils may promote TSC2-null cells at least in part via NE, to promote tumor cell actions.
Characterization of the human LAM tumor immune microenvironment is nascent, and the myeloid content of LAM lesions is understudied. The LAM Cell Atlas (Du et al. 2023) has identified various immune cell populations in the LAM lung, including tumor-perturbed neutrophils (Minor et al. 2023), as well as inflammatory macrophage and monocyte populations, conventional and plasmacytoid dendritic cells, and other granulocytes such as mast cells and basophils. While a few studies have suggested a potential role for macrophages and monocytes in the immune-suppressive LAM tumor immune microenvironment, as mentioned above, only mast cells and neutrophils have been assessed by functional assay, leaving much to be illuminated about the significance of the diverse myeloid compartment of the LAM tumor immune microenvironment. In addition, the well-documented immunomodulatory potential of estrogens, the robust estrogen sensitivity of LAM, and recent evidence that estradiol enhances tumor-induced expansion of neutrophils (Minor et al. 2023) highlight the need to integrate investigations related to the hormone sensitivity of LAM with those related to the tumor immune microenvironment. In short, it will be important to determine how estrogenic signaling contributes to the immune microenvironment of LAM lesions, as well as the metastatic progression of this disease.
Conclusions
In summary, the studies noted here, as well as many other outstanding discoveries in LAM that were not discussed due to space limitations, have helped researchers identify potential LAM biomarkers and treatment targets other than mTORC1 inhibition. Future studies will need to focus on understanding how the lung mesenchyme and the LAMCORE cells collaborate to mediate the unusual cystic phenotype in LAM, how melanocytic markers promote LAMCORE cell growth and also regulate the surrounding tumor immune microenvironment, and how estradiol may play a role in integrating all of these functions to help orchestrate LAM progression.
Declaration of interest
The authors have no disclosures.
Funding
SRH was supported by R01CA193583. BMNM was supported by T32AI007285 and F31CA254132. EG was supported by T32HL066988.
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