Senescent CAFs Mediate Immunosuppression and Drive Breast Cancer Progression.

The tumor microenvironment (TME) profoundly influences tumorigenesis, with gene expression in the breast TME capable of predicting clinical outcomes. The TME is complex and includes distinct cancer-associated fibroblast (CAF) subtypes whose contribution to tumorigenesis remains unclear. Here, we identify a subset of myofibroblast CAFs (myCAF) that are senescent (senCAF) in mouse and human breast tumors. Utilizing the MMTV-PyMT;INK-ATTAC (INK) mouse model, we found that senCAF-secreted extracellular matrix specifically limits natural killer (NK) cell cytotoxicity to promote tumor growth. Genetic or pharmacologic senCAF elimination unleashes NK cell killing, restricting tumor growth. Finally, we show that senCAFs are present in HER2+, ER+, and triple-negative breast cancer and in ductal carcinoma in situ (DCIS) where they predict tumor recurrence. Together, these findings demonstrate that senCAFs are potently tumor promoting and raise the possibility that targeting them by senolytic therapy could restrain breast cancer development. Significance: senCAFs limit NK cell-mediated killing, thereby contributing to breast cancer progression. Thus, targeting senCAFs could be a clinically viable approach to limit tumor progression. See related article by Belle et al., p. 1324.

FHB resistance conferred by Fhb1 is under inhibitory regulation of two genetic loci in wheat (Triticum aestivum L.).

KEY MESSAGE: Two loci inhibiting Fhb1 resistance to Fusarium head blight were identified through genome-wide association mapping and validated in biparental populations. Fhb1 confers Fusarium head blight (FHB) resistance by limiting fungal spread within spikes in wheat (type II resistance). However, not all lines with Fhb1 display the expected resistance. To identify genetic factors regulating Fhb1 effect, a genome-wide association study for type II resistance was first performed with 72 Fhb1-carrying lines using the Illumina 90 K iSelect SNP chip. Of 84 significant marker-trait associations detected, more than half were repeatedly detected in at least two environments, with the SNPs distributed in one region on chromosome 5B and one on chromosome 6A. This result was validated in a collection of 111 lines with Fhb1 and 301 lines without Fhb1. We found that these two loci caused significant resistance variations solely among lines with Fhb1 by compromising the resistance. In1, the inhibitory gene on chromosome 5B, was in close linkage with Xwgrb3860 in a recombinant inbred line population derived from Nanda2419 × Wangshuibai and a double haploid (DH) population derived from R-43 (Fhb1 near isogenic line) × Biansui7 (with Fhb1 and In1); and In2, the inhibitory gene on chromosome 6A, was mapped to the Xwgrb4113-Xwgrb4034 interval using a DH population derived from R-43 × PH8901 (with Fhb1 and In2). In1 and In2 are present in all wheat-growing areas worldwide. Their frequencies in China's modern cultivars are high but have significantly decreased in comparison with landraces. These findings are of great significance for FHB resistance breeding using Fhb1.

p38MAPKα Stromal Reprogramming Sensitizes Metastatic Breast Cancer to Immunotherapy.

Metastatic breast cancer is an intractable disease that responds poorly to immunotherapy. We show that p38MAPKα inhibition (p38i) limits tumor growth by reprogramming the metastatic tumor microenvironment in a CD4+ T cell-, IFNγ-, and macrophage-dependent manner. To identify targets that further increased p38i efficacy, we utilized a stromal labeling approach and single-cell RNA sequencing. Thus, we combined p38i and an OX40 agonist that synergistically reduced metastatic growth and increased overall survival. Intriguingly, patients with a p38i metastatic stromal signature had better overall survival that was further improved by the presence of an increased mutational load, leading us to ask if our approach would be effective in antigenic breast cancer. The combination of p38i, anti-OX40, and cytotoxic T-cell engagement cured mice of metastatic disease and produced long-term immunologic memory. Our findings demonstrate that a detailed understanding of the stromal compartment can be used to design effective antimetastatic therapies. SIGNIFICANCE: Immunotherapy is rarely effective in breast cancer. We dissected the metastatic tumor stroma, which revealed a novel therapeutic approach that targets the stromal p38MAPK pathway and creates an opportunity to unleash an immunologic response. Our work underscores the importance of understanding the tumor stromal compartment in therapeutic design. This article is highlighted in the In This Issue feature, p. 1275.

Therapy-Induced Senescence Drives Bone Loss.

Chemotherapy is important for cancer treatment, however, toxicities limit its use. While great strides have been made to ameliorate the acute toxicities induced by chemotherapy, long-term comorbidities including bone loss remain a significant problem. Chemotherapy-driven estrogen loss is postulated to drive bone loss, but significant data suggests the existence of an estrogen-independent mechanism of bone loss. Using clinically relevant mouse models, we showed that senescence and its senescence-associated secretory phenotype (SASP) contribute to chemotherapy-induced bone loss that can be rescued by depleting senescent cells. Chemotherapy-induced SASP could be limited by targeting the p38MAPK-MK2 pathway, which resulted in preservation of bone integrity in chemotherapy-treated mice. These results transform our understanding of chemotherapy-induced bone loss by identifying senescent cells as major drivers of bone loss and the p38MAPK-MK2 axis as a putative therapeutic target that can preserve bone and improve a cancer survivor's quality of life. SIGNIFICANCE: Senescence drives chemotherapy-induced bone loss that is rescued by p38MAPK or MK2 inhibitors. These findings may lead to treatments for therapy-induced bone loss, significantly increasing quality of life for cancer survivors.

Inhibition of the Stromal p38MAPK/MK2 Pathway Limits Breast Cancer Metastases and Chemotherapy-Induced Bone Loss.

The role of the stromal compartment in tumor progression is best illustrated in breast cancer bone metastases, where the stromal compartment supports tumor growth, albeit through poorly defined mechanisms. p38MAPKα is frequently expressed in tumor cells and surrounding stromal cells, and its expression levels correlate with poor prognosis. This observation led us to investigate whether inhibition of p38MAPKα could reduce breast cancer metastases in a clinically relevant model. Orally administered, small-molecule inhibitors of p38MAPKα or its downstream kinase MK2 each limited outgrowth of metastatic breast cancer cells in the bone and visceral organs. This effect was primarily mediated by inhibition of the p38MAPKα pathway within the stromal compartment. Beyond effectively limiting metastatic tumor growth, these inhibitors reduced tumor-associated and chemotherapy-induced bone loss, which is a devastating comorbidity that drastically affects quality of life for patients with cancer. These data underscore the vital role played by stromal-derived factors in tumor progression and identify the p38MAPK-MK2 pathway as a promising therapeutic target for metastatic disease and prevention of tumor-induced bone loss.Significance: Pharmacologically targeting the stromal p38MAPK-MK2 pathway limits metastatic breast cancer growth, preserves bone quality, and extends survival. Cancer Res; 78(19); 5618-30. ©2018 AACR.

Stromal senescence establishes an immunosuppressive microenvironment that drives tumorigenesis.

Age is a significant risk factor for the development of cancer. However, the mechanisms that drive age-related increases in cancer remain poorly understood. To determine if senescent stromal cells influence tumorigenesis, we develop a mouse model that mimics the aged skin microenvironment. Using this model, here we find that senescent stromal cells are sufficient to drive localized increases in suppressive myeloid cells that contributed to tumour promotion. Further, we find that the stromal-derived senescence-associated secretory phenotype factor interleukin-6 orchestrates both increases in suppressive myeloid cells and their ability to inhibit anti-tumour T-cell responses. Significantly, in aged, cancer-free individuals, we find similar increases in immune cells that also localize near senescent stromal cells. This work provides evidence that the accumulation of senescent stromal cells is sufficient to establish a tumour-permissive, chronic inflammatory microenvironment that can shelter incipient tumour cells, thus allowing them to proliferate and progress unabated by the immune system.

Stromal-Initiated Changes in the Bone Promote Metastatic Niche Development.

More than 85% of advanced breast cancer patients suffer from metastatic bone lesions, yet the mechanisms that facilitate these metastases remain poorly understood. Recent studies suggest that tumor-derived factors initiate changes within the tumor microenvironment to facilitate metastasis. However, whether stromal-initiated changes are sufficient to drive increased metastasis in the bone remains an open question. Thus, we developed a model to induce reactive senescent osteoblasts and found that they increased breast cancer colonization of the bone. Analysis of senescent osteoblasts revealed that they failed to mineralize bone matrix and increased local osteoclastogenesis, the latter process being driven by the senescence-associated secretory phenotype factor, IL-6. Neutralization of IL-6 was sufficient to limit senescence-induced osteoclastogenesis and tumor cell localization to bone, thereby reducing tumor burden. Together, these data suggest that a reactive stromal compartment can condition the niche, in the absence of tumor-derived signals, to facilitate metastatic tumor growth in the bone.

p38MAPK plays a crucial role in stromal-mediated tumorigenesis.

UNLABELLED: Neoplastic cells rely on the tumor microenvironment (TME) for survival and progression factors. Indeed, senescent and cancer-associated fibroblasts (CAF) express factors that promote tumorigenesis that are collectively referred to as the senescence-associated secretory phenotype (SASP). Despite their importance in tumorigenesis, the mechanisms that control TME-derived factor expression remain poorly understood. Here, we address a key unanswered question: how the SASP is sustained in senescent fibroblasts and CAFs. We find that the mitogen-activated protein kinase p38 (p38MAPK) controls AUF1 occupancy on SASP mRNAs and thus controls their stability. The importance of this regulatory mechanism is underscored by our findings that stromal-specific p38MAPK inhibition abrogates the tumor-promoting activities of CAFs and senescent fibroblasts. Our data suggest that targeting SASP mRNA stability through inhibition of p38MAPK will significantly aid the development of clinical strategies to target the TME. SIGNIFICANCE: The TME plays a key role in tumorigenesis. We demonstrate that p38MAPK governs a posttranscriptional mechanism that sustains the protumorigenic SASP. Inhibition of p38MAPK abrogates the tumor-promoting activities of CAFs and senescent fibroblasts. Thus, p38MAPK is a TME-specific Achilles' heel that may be exploited as a new therapeutic target.

Osteopontin stimulates preneoplastic cellular proliferation through activation of the MAPK pathway.

Alterations in the microenvironment collaborate with cell autonomous mutations during the transformation process. Indeed, cancer-associated fibroblasts and senescent fibroblasts stimulate tumorigenesis in xenograft models. Because senescent fibroblasts accumulate with age, these findings suggest that they contribute to age-related increases in tumorigenesis. Previously we showed that senescence-associated stromal-derived osteopontin contributes to preneoplastic cell growth in vitro and in xenografts, suggesting that it impacts neoplastic progression. Analysis of fibroblasts within premalignant and malignant skin lesions ranging from solar/actinic keratosis to squamous cell carcinoma revealed they express osteopontin. Given the stromal expression of osteopontin, we investigated how osteopontin impacts preneoplastic cell growth. We show that osteopontin promotes preneoplastic keratinocyte cellular proliferation and cell survival through the CD44 cell receptor and activation of the MAPK pathway. These data suggest that stromal-derived osteopontin impacts tumorigenesis by stimulating preneoplastic cell proliferation thus allowing expansion of initiated cells in early lesions.

Cadm1 expression and function in the mouse lens.

PURPOSE: The immunoglobulin superfamily member Cadm1 is a single-pass, type 1 membrane protein that mediates calcium-independent, cell-cell adhesion. Cadm1 has been implicated in tumor formation and synaptogenesis. A recent analysis of mouse lens cell membranes identified Cadm1 as a major constituent of the fiber cell membrane proteome. Here the authors examined the expression and function of Cadm1 in the mouse lens. METHODS: Cadm1 expression was analyzed by Western blotting and immunofluorescence. The morphology of individual wild-type and Cadm1-null lens cells was visualized by confocal microscopy. RESULTS: Cadm1 was present in epithelial and superficial fiber cells as a heavily glycosylated protein with an apparent molecular mass of ≈80 kDa. Analysis of proteins extracted from various strata of the lens indicated that Cadm1 was degraded during fiber cell differentiation, at approximately the same time as the lens organelles, an observation confirmed by confocal microscopy. In epithelial cells, Cadm1 was enriched in basolateral membranes, whereas, in fiber cells, expression was restricted to the lateral membranes. Lenses from Cadm1-null mice were of normal size and transparency. The three-dimensional morphology of the cells in the epithelial layer was unaltered in the absence of Cadm1. However, in contrast to wild-type lens fiber cells, Cadm1-null fiber cells had an irregular, highly undulating morphology. CONCLUSIONS: Cadm1 is an abundant component of the lens fiber cell membrane. Although not essential for lens transparency, Cadm1 has an indispensable role in establishing and maintaining the characteristic three-dimensional architecture of the lens fiber cell mass.

Senescent stromal-derived osteopontin promotes preneoplastic cell growth.

Alterations in the tissue microenvironment collaborate with cell autonomous genetic changes to contribute to neoplastic progression. The importance of the microenvironment in neoplastic progression is underscored by studies showing that fibroblasts isolated from a tumor stimulate the growth of preneoplastic and neoplastic cells in xenograft models. Similarly, senescent fibroblasts promote preneoplastic cell growth in vitro and in vivo. Because senescent cells accumulate with age, their presence is hypothesized to facilitate preneoplastic cell growth and tumor formation in older individuals. To identify senescent stromal factors directly responsible for stimulating preneoplastic cell growth, we carried out whole-genome transcriptional profiling and compared senescent fibroblasts with their younger counterparts. We identified osteopontin (OPN) as one of the most highly elevated transcripts in senescent fibroblasts. Importantly, reduction of OPN protein levels by RNA interference did not affect senescence induction in fibroblasts; however, it dramatically reduced the growth-promoting activities of senescent fibroblasts in vitro and in vivo, showing that OPN is necessary for paracrine stimulation of preneoplastic cell growth. In addition, we found that recombinant OPN was sufficient to stimulate preneoplastic cell growth. Finally, we show that OPN is expressed in senescent stroma within preneoplastic lesions that arise following 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate treatment of mice, suggesting that stromal-derived OPN-mediated signaling events affect neoplastic progression.

Lens fluorescence and accommodative amplitude in pre-presbyopic and presbyopic subjects.

Accommodative amplitude (AA; the difference, measured in diopters, between the near and far points of vision) declines steadily with age such that, by midlife, most individuals are unable to focus clearly on near objects and, thus, are said to be presbyopic. Conversely, intrinsic lens fluorescence (LF) increases steadily with age. Previous studies have suggested that AA and LF are negatively correlated, independent of age. Were this to be the case, it might suggest that the biochemical modifications underlying increased tissue fluorescence (for example, glycation of lens proteins) contribute to presbyopia. We used quantitative techniques to re-evaluate the relationship between AA and LF in 161 healthy volunteers aged between 25 and 70. Our data confirmed that AA decreases with age, becoming essentially zero by age 55, and LF increases with age. However, in marked contrast to previous reports, statistical analysis failed to detect any correlation between LF and AA independent of age. Thus, the biochemical processes responsible for increased LF observed in the aged lens are unlikely to contribute directly to presbyopia.

Susceptibilities to and mechanisms of excitotoxic cell death of adult mouse inner retinal neurons in dissociated culture.

PURPOSE: To explore the susceptibilities of adult retinal neurons in dissociated culture to treatments with excitotoxic agonists and the mechanisms of the resultant retinal cell death. METHODS: C57B6 mice were used. Retinas were removed, dissociated, plated on a polylysine/laminin substrate, and maintained in vitro for 5 to 7 days. Excitotoxic agonists (glutamate, N-methyl-D-aspartate [NMDA], or kainic acid [KA]) were added for 30 minutes or 24 hours, sometimes in the presence of modified extracellular ion concentrations or potential blocking agents. The next day, cells were fixed and immunocytochemically stained to identify ganglion and amacrine cells. Surviving cells were counted. RESULTS: Ganglion cells from adult mouse retinas were much less susceptible to excitotoxic death than those prepared from neonatal retinas. Adult amacrine cells were killed by KA, NMDA, or glutamate. Experiments with selective blockers demonstrated that KA killed through AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, whereas NMDA and glutamate exerted toxicity through a combination of AMPA and NMDA receptors. The KA-induced death of amacrine cells was not mediated by chloride ions. Removal of extracellular sodium, however, completely prevented the amacrine cell death, and removal of extracellular calcium prevented approximately 70% of the death. The path of calcium entry was investigated. Experiments with selective blockers indicated that the lethal calcium entry was via reverse operation of a sodium-calcium exchanger. CONCLUSIONS: There is a profound developmental regulation in the sensitivity of retina ganglion cells to excitotoxic insults. Excessive intracellular sodium and calcium are the proximal causes of amacrine cell death. The pathologic calcium entry is dependent on the sodium overload, which then drives a sodium-calcium exchanger to take up calcium.