MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib.

BACKGROUND: Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful. METHODS: To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cells. RESULTS: MCL-1 inhibition caused apoptosis of basal-like MDA-MB-468-2A cells grown as monolayers, and sensitized them to the BCL-2/BCL-XL inhibitor ABT-263, demonstrating that MCL-1 regulated cell survival. In MDA-MB-231-2A cells, grown in an organotypic model, induction of BIMs2A produced an almost complete suppression of invasion. Apoptosis was induced in such a small proportion of these cells that it could not account for the large decrease in invasion, suggesting that MCL-1 was operating via a previously undetected mechanism. MCL-1 antagonism also suppressed local invasion and distant metastasis to the lung in mouse mammary intraductal xenografts. Kinomic profiling revealed that MCL-1 antagonism modulated Src family kinases and their targets, which suggested that MCL-1 might act as an upstream modulator of invasion via this pathway. Inhibition of MCL-1 in combination with dasatinib suppressed invasion in 3D models of invasion and inhibited the establishment of tumors in vivo. CONCLUSION: These data provide the first evidence that MCL-1 drives breast cancer cell invasion and suggests that MCL-1 antagonists could be used alone or in combination with drugs targeting Src kinases such as dasatinib to suppress metastasis.

High mammographic density is associated with an increase in stromal collagen and immune cells within the mammary epithelium.

INTRODUCTION: Mammographic density (MD), after adjustment for a women's age and body mass index, is a strong and independent risk factor for breast cancer (BC). Although the BC risk attributable to increased MD is significant in healthy women, the biological basis of high mammographic density (HMD) causation and how it raises BC risk remain elusive. We assessed the histological and immunohistochemical differences between matched HMD and low mammographic density (LMD) breast tissues from healthy women to define which cell features may mediate the increased MD and MD-associated BC risk. METHODS: Tissues were obtained between 2008 and 2013 from 41 women undergoing prophylactic mastectomy because of their high BC risk profile. Tissue slices resected from the mastectomy specimens were X-rayed, then HMD and LMD regions were dissected based on radiological appearance. The histological composition, aromatase immunoreactivity, hormone receptor status and proliferation status were assessed, as were collagen amount and orientation, epithelial subsets and immune cell status. RESULTS: HMD tissue had a significantly greater proportion of stroma, collagen and epithelium, as well as less fat, than LMD tissue did. Second harmonic generation imaging demonstrated more organised stromal collagen in HMD tissues than in LMD tissues. There was significantly more aromatase immunoreactivity in both the stromal and glandular regions of HMD tissues than in those regions of LMD tissues, although no significant differences in levels of oestrogen receptor, progesterone receptor or Ki-67 expression were detected. The number of macrophages within the epithelium or stroma did not change; however, HMD stroma exhibited less CD206(+) alternatively activated macrophages. Epithelial cell maturation was not altered in HMD samples, and no evidence of epithelial-mesenchymal transition was seen; however, there was a significant increase in vimentin(+)/CD45(+) immune cells within the epithelial layer in HMD tissues. CONCLUSIONS: We confirmed increased proportions of stroma and epithelium, increased aromatase activity and no changes in hormone receptor or Ki-67 marker status in HMD tissue. The HMD region showed increased collagen deposition and organisation as well as decreased alternatively activated macrophages in the stroma. The HMD epithelium may be a site for local inflammation, as we observed a significant increase in CD45(+)/vimentin(+) immune cells in this area.

E-cadherin in developing murine T cells controls spindle alignment and progression through ß-selection.

A critical stage of T cell development is ß-selection; at this stage, the T cell receptor ß (TCRß) chain is generated, and the developing T cell starts to acquire antigenic specificity. Progression through ß-selection is assisted by low-affinity interactions between the nascent TCRß chain and peptide presented on stromal major histocompatibility complex and cues provided by the niche. In this study, we identify a cue within the developing T cell niche that is critical for T cell development. E-cadherin mediates cell-cell interactions and influences cell fate in many developmental systems. In developing T cells, E-cadherin contributed to the formation of an immunological synapse and the alignment of the mitotic spindle with the polarity axis during division, which facilitated subsequent T cell development. Collectively, these data suggest that E-cadherin facilitates interactions with the thymic niche to coordinate the ß-selection stage of T cell development.

TCF-1 limits intraepithelial lymphocyte antitumor immunity in colorectal carcinoma.

Intraepithelial lymphocytes (IELs), including αß and γδ T cells (T-IELs), constantly survey and play a critical role in maintaining the gastrointestinal epithelium. We show that cytotoxic molecules important for defense against cancer were highly expressed by T-IELs in the small intestine. In contrast, abundance of colonic T-IELs was dependent on the microbiome and displayed higher expression of TCF-1/TCF7 and a reduced effector and cytotoxic profile, including low expression of granzymes. Targeted deletion of TCF-1 in γδ T-IELs induced a distinct effector profile and reduced colon tumor formation in mice. In addition, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers (CRCs) compared with normal healthy colon, which strongly correlated with an enhanced γδ T-IEL effector phenotype and improved patient survival. Our work identifies TCF-1 as a colon-specific T-IEL transcriptional regulator that could inform new immunotherapy strategies to treat CRC.

Establishing a multiplex imaging panel to study T cell development in the thymus in mouse.

Multiplexed immunohistochemistry enables analysis of cellular and signaling events in the context of an intact organ. Here, we describe protocols for applying multiplexed immunohistochemistry to the mouse thymus. In particular, we describe how to identify cells at the specific differentiation stage known as ß-selection, and to monitor pre-TCR signaling and the cellular response at that stage. For complete details on the use and execution of this protocol, please refer to Allam et al. (2021).

Paracrine IL-6 Signaling Confers Proliferation between Heterogeneous Inflammatory Breast Cancer Sub-Clones.

Inflammatory breast cancer (IBC) describes a highly aggressive form of breast cancer of diverse molecular subtypes and clonal heterogeneity across individual tumors. Accordingly, IBC is recognized by its clinical signs of inflammation, associated with expression of interleukin (IL)-6 and other inflammatory cytokines. Here, we investigate whether sub-clonal differences between expression of components of the IL-6 signaling cascade reveal a novel role for IL-6 to mediate a proliferative response in trans using two prototypical IBC cell lines. We find that SUM149 and SUM 190 cells faithfully replicate differential expression observed in a subset of human IBC specimens between IL-6, the activated form of the key downstream transcription factor STAT3, and of the HER2 receptor. Surprisingly, the high level of IL-6 produced by SUM149 cells activates STAT3 and stimulates proliferation in SUM190 cells, but not in SUM149 cells with low IL-6R expression. Importantly, SUM149 conditioned medium or co-culture with SUM149 cells induced growth of SUM190 cells, and this effect was abrogated by the IL-6R neutralizing antibody Tocilizumab. The results suggest a novel function for inter-clonal IL-6 signaling in IBC, whereby IL-6 promotes in trans proliferation of IL-6R and HER2-expressing responsive sub-clones and, therefore, may provide a vulnerability that can be exploited therapeutically by repurposing of a clinically approved antibody.

Tumor Growth Remains Refractory to Myc Ablation in Host Macrophages.

Aberrant expression of the oncoprotein c-Myc (Myc) is frequently observed in solid tumors and is associated with reduced overall survival. In addition to well-recognized cancer cell-intrinsic roles of Myc, studies have also suggested tumor-promoting roles for Myc in cells of the tumor microenvironment, including macrophages and other myeloid cells. Here, we benchmark Myc inactivation in tumor cells against the contribution of its expression in myeloid cells of murine hosts that harbor endogenous or allograft tumors. Surprisingly, we observe that LysMCre-mediated Myc ablation in host macrophages does not attenuate tumor growth regardless of immunogenicity, the cellular origin of the tumor, the site it develops, or the stage along the tumor progression cascade. Likewise, we find no evidence for Myc ablation to revert or antagonize the polarization of alternatively activated immunosuppressive macrophages. Thus, we surmise that systemic targeting of Myc activity may confer therapeutic benefits primarily through limiting Myc activity in tumor cells rather than reinvigorating the anti-tumor activity of macrophages.

Developing T cells form an immunological synapse for passage through the ß-selection checkpoint.

The ß-selection checkpoint of T cell development tests whether the cell has recombined its genomic DNA to produce a functional T cell receptor ß (TCRß). Passage through the ß-selection checkpoint requires the nascent TCRß protein to mediate signaling through a pre-TCR complex. In this study, we show that developing T cells at the ß-selection checkpoint establish an immunological synapse in in vitro and in situ, resembling that of the mature T cell. The immunological synapse is dependent on two key signaling pathways known to be critical for the transition beyond the ß-selection checkpoint, Notch and CXCR4 signaling. In vitro and in situ analyses indicate that the immunological synapse promotes passage through the ß-selection checkpoint. Collectively, these data indicate that developing T cells regulate pre-TCR signaling through the formation of an immunological synapse. This signaling platform integrates cues from Notch, CXCR4, and MHC on the thymic stromal cell to allow transition beyond the ß-selection checkpoint.

Exploiting the STAT3 Nexus in Cancer-Associated Fibroblasts to Improve Cancer Therapy.

The tumor microenvironment (TME) is composed of a heterogenous population of cells that exist alongside the extracellular matrix and soluble components. These components can shape an environment that is conducive to tumor growth and metastatic spread. It is well-established that stromal cancer-associated fibroblasts (CAFs) in the TME play a pivotal role in creating and maintaining a growth-permissive environment for tumor cells. A growing body of work has uncovered that tumor cells recruit and educate CAFs to remodel the TME, however, the mechanisms by which this occurs remain incompletely understood. Recent studies suggest that the signal transducer and activator of transcription 3 (STAT3) is a key transcription factor that regulates the function of CAFs, and their crosstalk with tumor and immune cells within the TME. CAF-intrinsic STAT3 activity within the TME correlates with tumor progression, immune suppression and eventually the establishment of metastases. In this review, we will focus on the roles of STAT3 in regulating CAF function and their crosstalk with other cells constituting the TME and discuss the utility of targeting STAT3 within the TME for therapeutic benefit.

Re-exploration for bleeding after cardiac surgery: revaluation of urgency and factors promoting low rate.

BACKGROUND: Re-exploration of bleeding after cardiac surgery is associated with significant morbidity and mortality. Perioperative blood loss and rate of re-exploration are variable among centers and surgeons. OBJECTIVE: To present our experience of low rate of re-exploration based on adopting checklist for hemostasis and algorithm for management. METHODS: Retrospective analysis of medical records was conducted for 565 adult patients who underwent surgical treatment of congenital and acquired heart disease and were complicated by postoperative bleeding from Feb 2006 to May 2019. Demographics of patients, operative characteristics, perioperative risk factors, blood loss, requirements of blood transfusion, morbidity and mortality were recorded. Logistic regression was used to identify predictors of re-exploration and determinants of adverse outcome. RESULTS: Thirteen patients (1.14%) were reexplored for bleeding. An identifiable source of bleeding was found in 11 (84.6%) patients. Risk factors for re-exploration were high body mass index, high Euro SCORE, operative priority (urgent/emergent), elevated serum creatinine and low platelets count. Re-exploration was significantly associated with increased requirements of blood transfusion, adverse effects on cardiorespiratory state (low ejection fraction, increased s. lactate, and prolonged period of mechanical ventilation), longer intensive care unit stay, hospital stay, increased incidence of SWI, and higher mortality (15.4% versus 2.53% for non-reexplored patients). We managed 285 patients with severe or massive bleeding conservatively by hemostatic agents according to our protocol with no added risk of morbidity or mortality. CONCLUSION: Low rate of re-exploration for bleeding can be achieved by strict preoperative preparation, intraoperative checklist for hemostasis implemented by senior surgeons and adopting an algorithm for management.

Context-Specific Mechanisms of Cell Polarity Regulation.

Cell polarity is an essential process shared by almost all animal tissues. Moreover, cell polarity enables cells to sense and respond to the cues provided by the neighboring cells and the surrounding microenvironment. These responses play a critical role in regulating key physiological processes, including cell migration, proliferation, differentiation, vesicle trafficking and immune responses. The polarity protein complexes regulating these interactions are highly evolutionarily conserved between vertebrates and invertebrates. Interestingly, these polarity complexes interact with each other and key signaling pathways in a cell-polarity context-dependent manner. However, the exact mechanisms by which these interactions take place are poorly understood. In this review, we will focus on the roles of the key polarity complexes SCRIB, PAR and Crumbs in regulating different forms of cell polarity, including epithelial cell polarity, cell migration, asymmetric cell division and the T-cell immunological synapse assembly and signaling.

Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis.

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer.

A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.

Increased S-nitrosylation and proteasomal degradation of caspase-3 during infection contribute to the persistence of adherent invasive Escherichia coli (AIEC) in immune cells.

Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn's disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients.