TGF-ß promotes heterogeneity and drug resistance in squamous cell carcinoma.

Subsets of long-lived, tumor-initiating stem cells often escape cancer therapies. However, sources and mechanisms that generate tumor heterogeneity and drug-resistant cell population are still unfolding. Here, we devise a functional reporter system to lineage trace and/or genetic ablate signaling in TGF-ß-activated squamous cell carcinoma stem cells (SCC-SCs). Dissecting TGF-ß's impact on malignant progression, we demonstrate that TGF-ß concentrating near tumor-vasculature generates heterogeneity in TGF-ß signaling at tumor-stroma interface and bestows slower-cycling properties to neighboring SCC-SCs. While non-responding progenies proliferate faster and accelerate tumor growth, TGF-ß-responding progenies invade, aberrantly differentiate, and affect gene expression. Intriguingly, TGF-ß-responding SCC-SCs show increased protection against anti-cancer drugs, but slower-cycling alone does not confer survival. Rather, TGF-ß transcriptionally activates p21, which stabilizes NRF2, thereby markedly enhancing glutathione metabolism and diminishing effectiveness of anti-cancer therapeutics. Together, these findings establish a surprising non-genetic paradigm for TGF-ß signaling in fueling heterogeneity in SCC-SCs, tumor characteristics, and drug resistance.

An emerging role for cellular crosstalk in the cancer stem cell niche.

Although cumulative genetic and epigenetic changes in cancer cells are correlated with tumor malignancy, accumulating evidence supports that tumor cell-extrinsic mechanisms play an essential role in driving tumor progression. The tissue architecture surrounding tumor cells evolves during disease progression and becomes a significant barrier to cancer treatments. The functional traits of the tumor microenvironment (TME), either tumor suppressive or supportive, are defined by the distribution of various stromal cells and their sequential and reciprocal cellular interactions. Recent studies have uncovered a significant heterogeneity in stromal cells and identified specific subpopulations correlated with clinical outcomes, providing novel insights into the complex TME system that drives tumor progression and therapy resistance. Moreover, a small population of tumor cells with tumor-initiating and drug-resistant capabilities, cancer stem cells (CSCs), is maintained by the specialized TME, the so-called CSC niche. The crosstalk between CSCs and niche cells is an attractive avenue for identifying the vulnerability of difficult-to-treat cancers. Here, we review the recent advance in understanding TME biology and its impact on CSCs. We then focus on a newly identified niche signaling loop by which CSCs promote malignant progression and drug resistance of squamous cell carcinoma. The CSC niche is a promising research field that needs more attention and could facilitate the development of durable cancer treatment. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cancer stem cells and their niche in the progression of squamous cell carcinoma.

Most cancers harbor a small population of highly tumorigenic cells known as cancer stem cells (CSCs). Because of their stem cell-like properties and resistance to conventional therapies, CSCs are considered to be a rational target for curable cancer treatment. However, despite recent advances in CSC research, CSC-targeted therapies are not as successful as was initially hoped. The proliferative, invasive, and drug-resistant properties of CSCs are regulated by the tumor microenvironment associated with them, the so-called CSC niche. Thus, targeting tumor-promoting cellular crosstalk between CSCs and their niches is an attractive avenue for developing durable therapies. Using mouse models of squamous cell carcinoma (SCC), we have demonstrated that tumor cells responding to transforming growth factor ß (TGF-ß) function as drug-resistant CSCs. The gene expression signature of TGF-ß-responding tumor cells has accelerated the identification of novel pathways that drive invasive tumor progression. Moreover, by focusing on the cytokine milieu and macrophages in the proximity of TGF-ß-responding tumor cells, we recently uncovered the molecular basis of a CSC-niche interaction that emerges during early tumor development. This review article summarizes the specialized tumor microenvironment associated with CSCs and discusses mechanisms by which malignant properties of CSCs are maintained and promoted.

RNAi screens in mice identify physiological regulators of oncogenic growth.

Tissue growth is the multifaceted outcome of a cell's intrinsic capabilities and its interactions with the surrounding environment. Decoding these complexities is essential for understanding human development and tumorigenesis. Here we tackle this problem by carrying out the first genome-wide RNA-interference-mediated screens in mice. Focusing on skin development and oncogenic (Hras(G12V)-induced) hyperplasia, our screens uncover previously unknown as well as anticipated regulators of embryonic epidermal growth. Among the top oncogenic screen hits are Mllt6 and the Wnt effector ß-catenin, which maintain Hras(G12V)-dependent hyperproliferation. We also expose ß-catenin as an unanticipated antagonist of normal epidermal growth, functioning through Wnt-independent intercellular adhesion. Finally, we validate functional significance in mouse and human cancers, thereby establishing the feasibility of in vivo mammalian genome-wide investigations to dissect tissue development and tumorigenesis. By documenting some oncogenic growth regulators, we pave the way for future investigations of other hits and raise promise for unearthing new targets for cancer therapies.

An RNA interference screen uncovers a new molecule in stem cell self-renewal and long-term regeneration.

Adult stem cells sustain tissue maintenance and regeneration throughout the lifetime of an animal. These cells often reside in specific signalling niches that orchestrate the stem cell's balancing act between quiescence and cell-cycle re-entry based on the demand for tissue regeneration. How stem cells maintain their capacity to replenish themselves after tissue regeneration is poorly understood. Here we use RNA-interference-based loss-of-function screening as a powerful approach to uncover transcriptional regulators that govern the self-renewal capacity and regenerative potential of stem cells. Hair follicle stem cells provide an ideal model. These cells have been purified and characterized from their native niche in vivo and, in contrast to their rapidly dividing progeny, they can be maintained and passaged long-term in vitro. Focusing on the nuclear proteins and/or transcription factors that are enriched in stem cells compared with their progeny, we screened ∼2,000 short hairpin RNAs for their effect on long-term, but not short-term, stem cell self-renewal in vitro. To address the physiological relevance of our findings, we selected one candidate that was uncovered in the screen: TBX1. This transcription factor is expressed in many tissues but has not been studied in the context of stem cell biology. By conditionally ablating Tbx1 in vivo, we showed that during homeostasis, tissue regeneration occurs normally but is markedly delayed. We then devised an in vivo assay for stem cell replenishment and found that when challenged with repetitive rounds of regeneration, the Tbx1-deficient stem cell niche becomes progressively depleted. Addressing the mechanism of TBX1 action, we discovered that TBX1 acts as an intrinsic rheostat of BMP signalling: it is a gatekeeper that governs the transition between stem cell quiescence and proliferation in hair follicles. Our results validate the RNA interference screen and underscore its power in unearthing new molecules that govern stem cell self-renewal and tissue-regenerative potential.

Cep72 regulates the localization of key centrosomal proteins and proper bipolar spindle formation.

Microtubule-nucleation activity and structural integrity of the centrosome are critical for various cellular functions. The gamma-tubulin ring complexes (gammaTuRCs) localizing to the pericentriolar matrix (PCM) of the centrosome are major sites of microtubule nucleation. The PCM is thought to be created by two cognate large coiled-coil proteins, pericentrin/kendrin and CG-NAP/AKAP450, and its stabilization by Kizuna is essential for bipolar spindle formation. However, the mechanisms by which these proteins are recruited and organized into a proper structure with microtubule-organizing activity are poorly understood. Here we identify a centrosomal protein Cep72 as a Kizuna-interacting protein. Interestingly, Cep72 is essential for the localization of CG-NAP and Kizuna. Cep72 is also involved in gammaTuRC recruitment to the centrosome and CG-NAP confers the microtubule-nucleation activity on the gammaTuRCs. During mitosis, Cep72-mediated microtubule organization is important for converging spindle microtubules to the centrosomes, which is needed for chromosome alignment and tension generation between kinetochores. Our findings show that Cep72 is the key protein essential for maintaining microtubule-organizing activity and structural integrity of the centrosome.

The Plk1 target Kizuna stabilizes mitotic centrosomes to ensure spindle bipolarity.

Formation of a bipolar spindle is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, defects in centrosome number and structural organization can lead to a loss of bipolarity. In addition, microtubule-mediated pulling and pushing forces acting on centrosomes and chromosomes are also important for bipolar spindle formation. Polo-like kinase 1 (Plk1) is a highly conserved Ser/Thr kinase that has essential roles in the formation of a bipolar spindle with focused poles. However, the mechanism by which Plk1 regulates spindle-pole formation is poorly understood. Here, we identify a novel centrosomal substrate of Plk1, Kizuna (Kiz), depletion of which causes fragmentation and dissociation of the pericentriolar material from centrioles at prometaphase, resulting in multipolar spindles. We demonstrate that Kiz is critical for establishing a robust mitotic centrosome architecture that can endure the forces that converge on the centrosomes during spindle formation, and suggest that Plk1 maintains the integrity of the spindle poles by phosphorylating Kiz.

Cancer stem cell-derived extracellular vesicles preferentially target MHC-II-macrophages and PD1+ T cells in the tumor microenvironment.

Immunotherapy is an approved treatment option for head and neck squamous cell carcinoma (HNSCC). However, the response rate to immune checkpoint blockade is only 13% for recurrent HNSCC, highlighting the urgent need to better understand tumor-immune interplay, with the ultimate goal of improving patient outcomes. HNSCC present high local recurrence rates and therapy resistance that can be attributed to the presence of cancer stem cells (CSC) within tumors. CSC exhibit singular properties that enable them to avoid immune detection and eradication. How CSC communicate with immune cells and which immune cell types are preferentially found within the CSC niche are still open questions. Here, we used genetic approaches to specifically label CSC-derived extracellular vesicles (EVs) and to perform Sortase-mediated in vivo proximity labeling of CSC niche cells. We identified specific immune cell subsets that were selectively targeted by EVCSC and that were found in the CSC niche. Native EVCSC preferentially targeted MHC-II-macrophages and PD1+ T cells in the tumor microenvironment, which were the same immune cell subsets enriched within the CSC niche. These observations indicate that the use of genetic technologies able to track EVs without in vitro isolation are a valuable tool to unveil the biology of native EVCSC.

A mechanistic basis for the malignant progression of salivary gland tumors.

Salivary gland tumors are diverse neoplasms, likely reflecting differences in the tissue- and cell-of-origin. 80%-90% of tumors arising in the sublingual gland (SLG) are malignant, whereas the other major glands often form benign tumors. Owing to the lack of experimental models to explore the etiology of salivary gland tumors, the cellular and molecular bases of malignancy remain unknown. Here, we generated a murine model of HRASG12V-driven salivary gland tumors amenable to examine tumor onset and malignant progression. We found that HMGA2 marks the tumor onset, and transformed-SOX2+ stem/progenitor cells expand exclusively in SLG tumors. Lineage tracing experiments showed that SLG tumor cells undergo an extensive epithelial-mesenchymal transition (EMT) and TGF-ß-responding tumor cells are a source of mesenchymal tumor cells invading the surrounding stroma. This study advances our understanding of the mechanistic basis of salivary gland malignancy and may help combat this highly heterogeneous cancer.

Response to Comment on "Tumor-initiating cells establish an IL-33-TGF-ß niche signaling loop to promote cancer progression".

Kamphuis et al argue that macrophages accumulated in the proximity of tumor-initiating cells do not express the high-affinity immunoglobulin E receptor FcεRIα. Although we cannot exclude the possibility of nonspecific binding of anti-FcεRIα antibody (clone MAR-1), we provide evidence that macrophages in squamous cell carcinomas express FcεRIα and that IL-33 induces FcεRIα expression in bone marrow cell-derived macrophages.

Mitogen-activated protein kinase-activated kinase RSK2 plays a role in innate immune responses to influenza virus infection.

Viral infections induce signaling pathways in mammalian cells that stimulate innate immune responses and affect cellular processes, such as apoptosis, mitosis, and differentiation. Here, we report that the ribosomal protein S6 kinase alpha 3 (RSK2), which is activated through the "classical" mitogen-activated protein kinase pathway, plays a role in innate immune responses to influenza virus infection. RSK2 functions in the regulation of cell growth and differentiation but was not known to play a role in the cellular antiviral response. We have found that knockdown of RSK2 enhanced viral polymerase activity and growth of influenza viruses. Influenza virus infection stimulates NK-kappaB- and beta interferon-dependent promoters. This stimulation was reduced in RSK2 knockdown cells, suggesting that RSK2 executes its effect through innate immune response pathways. Furthermore, RSK2 knockdown suppressed influenza virus-induced phosphorylation of the double-stranded RNA-activated protein kinase PKR, a known antiviral protein. These findings establish a role for RSK2 in the cellular antiviral response.

Tumor-initiating cells establish an IL-33-TGF-ß niche signaling loop to promote cancer progression.

Targeting the cross-talk between tumor-initiating cells (TICs) and the niche microenvironment is an attractive avenue for cancer therapy. We show here, using a mouse model of squamous cell carcinoma, that TICs play a crucial role in creating a niche microenvironment that is required for tumor progression and drug resistance. Antioxidant activity in TICs, mediated by the transcription factor NRF2, facilitates the release of a nuclear cytokine, interleukin-33 (IL-33). This cytokine promotes differentiation of macrophages that express the high-affinity immunoglobulin E receptor FcεRIα and are in close proximity to TICs. In turn, these IL-33-responding FcεRIα+ macrophages send paracrine transforming growth factor ß (TGF-ß) signals to TICs, inducing invasive and drug-resistant properties and further upregulating IL-33 expression. This TIC-driven, IL-33-TGF-ß feedforward loop could potentially be exploited for cancer treatment.

ADAP1 promotes invasive squamous cell carcinoma progression and predicts patient survival.

Invasive squamous cell carcinoma (SCC) is aggressive cancer with a high risk of recurrence and metastasis, but the critical determinants of its progression remain elusive. Here, we identify ADAP1, a GTPase-activating protein (GAP) for ARF6 up-regulated in TGF-ß-responding invasive tumor cells, as a strong predictor of poor survival in early-stage SCC patients. Using a mouse model of SCC, we show that ADAP1 overexpression promotes invasive tumor progression by facilitating cell migration and breakdown of the basement membrane. We found that ADAP1-rich, TGF-ß-responding tumor cells exhibit cytoplasmic laminin localization, which correlated with the absence of laminin and type IV collagen from the pericellular basement membrane. Interestingly, although tumors overexpressing a GAP activity-deficient mutant of ADAP1 resulted in morphologically complex tumors, those tumor cells failed to breach the basement membrane. Moreover, Adap1 deletion in tumor cells ameliorated the basement membrane breakdown and had less invading cells in the stroma. Our study demonstrates that ADAP1 is a critical mediator of TGF-ß-induced cancer invasion and might be exploited for the treatment of high-risk SCC.

Human mutant huntingtin disrupts vocal learning in transgenic songbirds.

Speech and vocal impairments characterize many neurological disorders. However, the neurogenetic mechanisms of these disorders are not well understood, and current animal models do not have the necessary circuitry to recapitulate vocal learning deficits. We developed germline transgenic songbirds, zebra finches (Taneiopygia guttata) expressing human mutant huntingtin (mHTT), a protein responsible for the progressive deterioration of motor and cognitive function in Huntington's disease (HD). Although generally healthy, the mutant songbirds had severe vocal disorders, including poor vocal imitation, stuttering, and progressive syntax and syllable degradation. Their song abnormalities were associated with HD-related neuropathology and dysfunction of the cortical-basal ganglia (CBG) song circuit. These transgenics are, to the best of our knowledge, the first experimentally created, functional mutant songbirds. Their progressive and quantifiable vocal disorder, combined with circuit dysfunction in the CBG song system, offers a model for genetic manipulation and the development of therapeutic strategies for CBG-related vocal and motor disorders.

The harmonies played by TGF-ß in stem cell biology.

To rejuvenate tissues and/or repair wounds, stem cells must receive extrinsic signals from their surrounding environment and integrate them with their intrinsic abilities to self-renew and differentiate to make tissues. Increasing evidence suggests that the superfamily of transforming growth factor-ßs (TGF-ßs) constitute integral components in the intercellular crosstalk between stem cells and their microenvironment. In this review, we summarize recent advances in our understanding of TGF-ß superfamily functions in embryonic and adult stem cells. We discuss how these pathways help to define the physiological environment where stem cells reside, and how perturbations in the signaling circuitry contribute to cancers.

Paracrine TGF-ß signaling counterbalances BMP-mediated repression in hair follicle stem cell activation.

Hair follicle (HF) regeneration begins when communication between quiescent epithelial stem cells (SCs) and underlying mesenchymal dermal papillae (DP) generates sufficient activating cues to overcome repressive BMP signals from surrounding niche cells. Here, we uncover a hitherto unrecognized DP transmitter, TGF-ß2, which activates Smad2/3 transiently in HFSCs concomitant with entry into tissue regeneration. This signaling is critical: HFSCs that cannot sense TGF-ß exhibit significant delays in HF regeneration, whereas exogenous TGF-ß2 stimulates HFSCs in vivo and in vitro. By engineering TGF-ß- and BMP-reporter mice, we show that TGF-ß2 signaling antagonizes BMP signaling in HFSCs but not through competition for limiting Smad4-coactivator. Rather, our microarray, molecular, and genetic studies unveil Tmeff1 as a direct TGF-ß2/Smad2/3 target gene, expressed by activated HFSCs and physiologically relevant in restricting and lowering BMP thresholds in the niche. Connecting BMP activity to an SC's response to TGF-ßs may explain why these signaling factors wield such diverse cellular effects.