It is incompletely understood which factors in patients with multiple sclerosis (MS) act as a trigger for the immune system to attack the brain and spinal cord.
A potential factor is described by a research team in the journal Proceedings of the National Academy of Sciences, PNAS.
The medical researchers used an animal model to show that the protein Smad7 mobilizes immune cells in the intestines which, in turn, trigger inflammation in the central nervous system.
Analyses of intestinal tissue samples taken from MS patients confirmed the results, which were published online on 4 December 2019.
The study was conducted at the Department of Neurology and the Centre of Neuroimmunology at St. Josef-Hospital, university hospital of Ruhr-Universität Bochum.
The Bochum-based group with biologist Dr. Steffen Haupeltshofer and neurologists Professor Simon Faissner and Professor Ingo Kleiter, formerly at the Bochum university hospital, currently at Marianne-Strauß-Klinik in Berg, collaborated with other colleagues from Bochum, Bremen, Mainz, Düsseldorf, Jülich and Rome.
Protein Smad7 activates immune cells in the intestines
The research team initially analysed the signal protein Smad7 in intestinal immune cells in mice, or more precisely: in T-cells.
The researchers compared genetically modified mice with a normal and those with a particularly high quantity of Smad7 in T-cells as well as mice without any Smad7 in T-cells.
They monitored if the animals developed opticospinal encephalomyelitis – a disease that mimics MS in humans.
In addition, an abnormal ratio of regulatory to pathogenic mechanisms was identified in intestinal mucosa samples in patients.
The strongest clinical MS-like symptoms occurred in animals with an increased Smad7 level in T-cells. In their intestines, T-cells were more frequently activated, which then migrated into the central nervous system where they triggered inflammation.
Moreover, the ratio of protective regulatory T-cells to pathogenic autoreactive T-cells had changed. In mice that didn’t have any Smad7 protein, no clinical signs of a MS-like disease occurred.
Results confirmed using tissue samples from patients
In the next step, the researchers analysed tissue samples taken from the intestines of 27 MS patients and compared them with samples taken from 27 healthy individuals.
In the patients, they identified changes similar to those in the animal model: the signal protein Smad7 occurred more frequently in intestinal mucosa samples of MS patients than in those of healthy individuals; in addition, an abnormal ratio of regulatory to pathogenic mechanisms was identified in intestinal mucosa samples in patients.
“For other autoimmune diseases such as Crohn’s and other inflammatory bowel diseases, researchers are already aware that Smad7 offers a promising therapeutic target; our results suggest that the same is true for multiple sclerosis,” says Ingo Kleiter. “Researchers are increasingly exploring intestinal involvement in the development and progression of MS,” adds Simon Faissner.
Smad7, a negative regulator of TGF-β signaling, has been implicated in the pathogenesis and treatment of inflammatory bowel diseases (IBDs), including Crohn’s disease (CD) and ulcerative colitis (UC). Here, we found that Smad7 mediates intestinal inflammation by limiting the PDL2/1-PD1 axis in dendritic cells (DCs) and CD4+T cells.
Smad7 deficiency in DCs promotes TGF-β responsiveness and the co-inhibitory molecules PDL2/1 on DCs, and it further imprints T cell-PD1 signaling to promote Treg differentiation. DC-specific Smad7 deletion mitigates DSS-induced colitis by inducing CD103+PDL2/1+DCs and Tregs.
In addition, Smad7 deficiency in CD4+T cells promotes PD1 and PD1-induced Tregs in vitro.
The transfer of Smad7-deficient CD4+T cells enhances Tregs in vivo and protects against T cell-mediated colitis. Furthermore, Smad7 antisense ameliorates DSS-induced UC, increasing TGF-β and PDL2/1-PD1 signaling. Enhancing PD1 signaling directly via Fc-fused PDL2/1 is also beneficial. Our results identify how Smad7 mediates intestinal inflammation and leverages these pathways therapeutically, providing additional strategies for IBD intervention.
The development of metastatic melanoma is thought to require the dynamic shifting of neoplastic cells between proliferative and invasive phenotypes. Contrary to this conventional “phenotype switching” model, we now show that disease progression can involve malignant melanoma cells simultaneously displaying proliferative and invasive properties.
Using a genetic mouse model of melanoma in combination with in vitro analyses of melanoma cell lines, we found that conditional deletion of the downstream signaling molecule Smad4, which abrogates all canonical TGF-β signaling, indeed inhibited both tumor growth and metastasis. Conditional deletion of the inhibitory signaling factor Smad7, however, generated cells that are both highly invasive and proliferative, indicating that invasiveness is compatible with a high proliferation rate.
In fact, conditional Smad7 deletion led to sustained melanoma growth and at the same time promoted massive metastasis formation, a result consistent with data indicating that low SMAD7 levels in patient tumors are associated with a poor survival. Our findings reveal that modulation of SMAD7 levels can overcome the need for phenotype switching during tumor progression and may thus represent a therapeutic target in metastatic disease.
Despite recent progress in therapy, melanoma has remained by far the deadliest skin cancer, with a 5-year survival rate of only 15% to 20% (1). There is increasing evidence that the aggressiveness of the disease is largely due to an intrinsic plasticity of melanoma cells, allowing the dynamic and reversible switching from a high-proliferative/low-invasive to a low-proliferative/high-invasive state (2–5).
This so-called “phenotype switching” has been functionally associated with metastasis formation and therapy resistance (6–8, 5). Furthermore, phenotype switching has been linked to a shift between a transcriptional program governed by high expression of the microphthalmia-associated transcription factor (MITF), a melanoma lineage-survival oncogene, and a transcriptional program associated with high expression of the receptor tyrosine kinase AXL, a marker for resistance to various targeted therapies (9–12).
In contrast to proliferative melanoma cells characterized by high MITF expression, MITFlo cells with high-invasive properties show a G1 cell cycle arrest (13).
However, while the distinction between MITFloAXLhi and MITFhiAXLlo phenotypes was used to classify melanomas at the bulk tumor level (14), single-cell RNA-Seq of human melanoma samples has recently led to the identification of a small fraction of double-positive MITFhiAXLhi cells. It is conceivable that such double-high cells simply represent a transition stage between the 2 phenotypes, but alternatively, they may also have specific tumorigenic properties that are yet to be elucidated (15).
Phenotype switching has also been linked to tumor progression in several epithelial tumors (16, 17).
In these cases, a reversible epithelial-to-mesenchymal transition (EMT) promotes invasiveness and stem cell–like features in cancer cells (18), which are driven by a network of embryonic EMT-inducing transcription factors (EMT-TFs) of the SNAIL, TWIST, ZEB, and bHLH/E47 protein families (19).
Although melanoma cells are not derived from epithelial cells, they nevertheless show EMT-like processes that can be observed in culture and that have been associated with metastatic spread of the disease (20).
In particular, activation of the MAPK pathway by oncogenic BRAF or NRAS promotes a switch from SNAIL2/ZEB2 expression to TWIST1/ZEB1 expression, which enhances invasiveness of melanoma cells (21).
One of the major candidate pathways for driving reversible phenotype switching is SMAD-dependent TGF-β superfamily signaling, which controls EMT in many cancers (22, 23). Sustained expression of EMT-TFs in breast cancer cells is directly regulated by autocrine TGF-β signaling (24). In melanoma, several TGF-β isoforms and NODAL as well as different BMP ligands were shown to be expressed by tumor cells and to promote invasiveness in cell culture or in organotypic human skin cultures (6, 7, 25–27).
In support of this, attenuation of TGF-β signaling by overexpression of SMAD7, an inhibitory SMAD protein (28), or by treatment with a small molecule inhibitor reduced bone metastasis formation from cells injected into the cardiovascular system in immunocompromised mice (29, 30).
However, other studies reported that melanoma cells exhibit partial resistance to the antiproliferative activity of TGF-β family factors (31).
Moreover, TGF-β–dependent SMAD signaling and transcription were not restricted to invasive cells, but were also observed in proliferative human melanoma cells (31).
Likewise, in human tissue samples, nuclear pSMAD2/3, which mediates canonical TGF-β signaling, was detected at all stages of the disease, including in benign hyperplastic lesions and cutaneous primary melanoma as well as in invasive melanoma (32).
Finally, inhibition of SMAD2/3 signaling by SMAD7 overexpression not only affected the invasiveness of melanoma cells, but also reduced their capacity to grow in vitro and upon transplantation into immunocompromised mice (29).
The combined data suggest that SMAD-mediated signaling may exert various functions in melanoma, which are likely influenced by the cellular context and the TGF-β superfamily ligands the tumor cells are exposed to (23).
Therefore, in an attempt to mimic the tumor microenvironment with respect to TGF-β–dependent SMAD signaling, we treated melanoma cells with various combinations of TGF-β family factors and addressed the relevance of overall TGF-β/SMAD signaling in melanoma in vivo by means of a genetic mouse model, in which tumors develop spontaneously within an undisturbed 3D environment.
Searching for factors modulating the proliferative and invasive capacities of melanoma cells, we identified SMAD7 as a repressor of a transcriptional program associated with concomitant MITFhiAXLhi expression. In fact, conditional Smad7 deletion in vivo resulted in the emergence of a MITFhiAXLhi subpopulation of cells that were simultaneously proliferating and invasive and associated with increased macrometastasis formation.
These experiments identify integrated SMAD signaling as a key driver of melanoma initiation, growth, and metastatic progression, pointing to a new therapeutic vulnerability in melanoma.
Simon Faissner – RUB
The image is in the public domain.
Original Research: Closed access
“Smad7 in intestinal CD4+ T cells determines autoimmunity in a spontaneous model of multiple sclerosis”. Steffen Haupeltshofer, Teresa Leichsenring, Sarah Berg, Xiomara Pedreiturria, Stephanie C. Joachim, Iris Tischoff, Jan-Michel Otte, Tobias Bopp, Massimo C. Fantini, Charlotte Esser, Dieter Willbold, Ralf Gold, Simon Faissner, and Ingo Kleiter.