Amr H. Allam

Intraepithelial lymphocytes (IELs), including αβ 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.

A critical stage of T cell development is β-selection; at this stage the TCRβ chain is generated and the developing T cell starts to acquire antigenic specificity. Progression through β-selection is assisted by a low affinity interaction between the nascent TCRβ chain and peptide presented on stromal MHC and external cues provided by the niche, including Notch and CXCR4. In this study, we reveal the importance of a new cue within the murine developing T cell niche which 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 highlight a new aspect of the developing T cell niche and provide insights into the role of E-cadherin in the β-selection stage of T cell development.

The β-selection checkpoint of T cell development tests whether the cell has recombined its genomic DNA to produce a functional T cell receptor β (TCRβ). Passage through the β-selection checkpoint requires the nascent TCRβ protein to mediate signaling through a pre-TCR complex. In this study, we show that developing T cells at the β-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 β-selection checkpoint, Notch and CXCR4 signaling. In vitro and in situ analyses indicate that the immunological synapse promotes passage through the β-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 β-selection checkpoint.

The β-selection checkpoint of T cell development tests whether the cell has recombined its genomic DNA to produce a functional T Cell Receptor β (TCRβ) receptor. Passage through the β-selection checkpoint requires the nascent TCRβ protein to mediate signaling through a pre-TCR complex. In this study, we show that developing T cells at the β-selection checkpoint establish an immunological synapse in in vitro & 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 β-selection checkpoint, Notch and CXCR4 signaling. In vitro and in situ analyses indicate that the immunological synapse promotes passage through the β-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 β-selection checkpoint. Summary: T cell development requires testing whether genomic rearrangement has produced a T cell receptor capable of transmitting signals. Most T cells fail this test. Here, we show that passage through the β-selection checkpoint requires assembly of a platform to support TCR signaling.

E-cadherin and β-catenin are key proteins that are essential in the formation of the epithelial cell layer in the colon but their regulatory pathways that are disrupted in cancer metastasis are not completely understood. Mutated in colorectal cancer (MCC) is a tumour suppressor gene that is silenced by promoter methylation in colorectal cancer and particularly in patients with increased lymph node metastasis. Here, we show that MCC methylation is found in 45% of colon and 24% of rectal cancers and is associated with proximal colon, poorly differentiated, circumferential and mucinous tumours as well as increasing T stage and larger tumour size. Knockdown of MCC in HCT116 colon cancer cells caused a reduction in E-cadherin protein level, which is a hallmark of epithelial–mesenchymal transition in cancer, and consequently diminished the E-cadherin/β-catenin complex. MCC knockdown disrupted cell–cell adhesive strength and integrity in the dispase and transepithelial electrical resistance assays, enhanced hepatocyte growth factor-induced cell scatter and increased tumour cell invasiveness in an organotypic assay. The Src/Abl inhibitor dasatinib, a candidate anti-invasive drug, abrogated the invasive properties induced by MCC deficiency. Mechanistically, we establish that MCC interacts with the E-cadherin/β-catenin complex. These data provide a significant advance in the current understanding of cell–cell adhesion in colon cancer cells.

Fine-tuned manipulation of tumor tension and vasculature enhances response to chemotherapy and impairs metastatic spread in pancreatic cancer.

Pancreatic cancer has a devastating prognosis, with an overall 5-year survival rate of ~8%, restricted treatment options and characteristic molecular heterogeneity. SerpinB2 expression, particularly in the stromal compartment, is associated with reduced metastasis and prolonged survival in pancreatic ductal adenocarcinoma (PDAC) and our genomic analysis revealed that SERPINB2 is frequently deleted in PDAC. We show that SerpinB2 is required by stromal cells for normal collagen remodelling in vitro, regulating fibroblast interaction and engagement with collagen in the contracting matrix. In a pancreatic cancer allograft model, co-injection of PDAC cancer cells and SerpinB2^−/− mouse embryonic fibroblasts (MEFs) resulted in increased tumour growth, aberrant remodelling of the extracellular matrix (ECM) and increased local invasion from the primary tumour. These tumours also displayed elevated proteolytic activity of the primary biochemical target of SerpinB2—urokinase plasminogen activator (uPA). In a large cohort of patients with resected PDAC, we show that increasing uPA mRNA expression was significantly associated with poorer survival following pancreatectomy. This study establishes a novel role for SerpinB2 in the stromal compartment in PDAC invasion through regulation of stromal remodelling and highlights the SerpinB2/uPA axis for further investigation as a potential therapeutic target in pancreatic cancer.

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. 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. 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. 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.