Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders, where epithelial defects drive, at least in part, some of the pathology. We reconstituted human intestinal epithelial organ, by using three-dimension culture of human colon organoids. Our aim was to characterize morphological and functional phenotypes of control (non-IBD) organoids, compared to inflamed organoids from IBD patients. The results generated describe the epithelial defects associated with IBD in primary organoid cultures, and evaluate the use of this model for pharmacological testing of anti-inflammatory approaches. Human colonic tissues were obtained from either surgical resections or biopsies, all harvested in non-inflammatory zones. Crypts were isolated from controls (non-IBD) and IBD patients and were cultured up to 12-days. Morphological (size, budding formation, polarization, luminal content), cell composition (proliferation, differentiation, immaturity markers expression), and functional (chemokine and tight junction protein expression) parameters were measured by immunohistochemistry, RT-qPCR or western-blot. The effects of inflammatory cocktail or anti-inflammatory treatments were studied in controls and IBD organoid cultures respectively. Organoid cultures from controls or IBD patients had the same cell composition after 10 to 12-days of culture, but IBD organoid cultures showed an inflammatory phenotype with decreased size and budding capacity, increased cell death, luminal debris, and inverted polarization. Tight junction proteins were also significantly decreased in IBD organoid cultures. Inflammatory cytokine cocktail reproduced this inflammatory phenotype in non-IBD organoids. Clinically used treatments (5-ASA, glucocorticoids, anti-TNF) reduced some, but not all parameters. Inflammatory phenotype is associated with IBD epithelium, and can be studied in organoid cultures. This model constitutes a reliable human pre-clinical model to investigate new strategies targeting epithelial repair.

Somatic mutations within the epidermal growth factor receptor (EGFR) kinase domain are detected in 10% to 30% of human non–small cell lung cancers and are correlated with striking clinical responses in a subset of patients treated with EGFR kinase inhibitors, such as gefitinib and erlotinib. Cell-based studies suggest that these mutant EGFRs promote increased autophosphorylating activity on a subset of EGFR COOH-terminal tyrosines and the consequent engagement of a subset of downstream effectors. Because EGFR function is regulated at multiple levels in vivo, and it is therefore difficult to assess the direct consequences of these mutations on EGFR enzyme function, we measured EGFR catalytic activity in in vitro kinase assays using purified recombinant proteins corresponding to the cytoplasmic domain of wild-type and two frequently detected EGFR mutants (DelL747-P753insS and L858R). Both mutants exhibit substantially increased autophosphorylating activity relative to wild-type EGFR, and they exhibit distinct reaction kinetics. In addition, the mutant kinases are more sensitive to kinase inhibition by gefitinib, which seems to reflect their increased drug affinity. These findings suggest that the altered signaling properties and drug sensitivity of these EGFR mutants that have been observed in vivo largely result from differences in the catalytic properties of the kinase. In addition, we find that the T790M secondary “drug resistance mutation” of EGFR, which frequently arises in relapsed patients that initially responded to treatment, confers enhanced kinase activity to primary activating EGFR alleles and may, therefore, be oncogenic in some contexts. [Cancer Res 2007;67(5):2325–30]

Phosphatidylinositol 3‐kinase (PI3K) regulates many cellular functions including growth and survival, and its excessive activation is a hallmark of cancer. Somatostatin, acting through its G protein‐coupled receptor (GPCR) sst2, has potent proapoptotic and anti‐invasive activities on normal and cancer cells. Here, we report a novel mechanism for inhibiting PI3K activity. Somatostatin, acting through sst2, inhibits PI3K activity by disrupting a pre‐existing complex comprising the sst2 receptor and the p85 PI3K regulatory subunit. Surface plasmon resonance and molecular modeling identified the phosphorylated‐Y⁷¹ residue of a p85‐binding pYXXM motif in the first sst2 intracellular loop, and p85 COOH‐terminal SH2 as direct interacting domains. Somatostatin‐mediated dissociation of this complex as well as p85 tyrosine dephosphorylation correlates with sst2 tyrosine dephosphorylation on the Y⁷¹ residue. Mutating sst2‐Y⁷¹ disabled sst2 to interact with p85 and somatostatin to inhibit PI3K, consequently abrogating sst2's ability to suppress cell survival and tumor growth. These results provide the first demonstration of a physical interaction between a GPCR and p85, revealing a novel mechanism for negative regulation by ligand‐activated GPCR of PI3K‐dependent survival pathways, which may be an important molecular target for antineoplastic therapy.

The G protein-coupled sst2 somatostatin receptor acts as a negative cell growth regulator. Sst2 transmits antimitogenic signaling by recruiting and activating the tyrosine phosphatase SHP-1. We now identified Src and SHP-2 as sst2-associated molecules and demonstrated their role in sst2 signaling. Surface plasmon resonance and mutation analyses revealed that SHP-2 directly associated with phosphorylated tyrosine 228 and 312, which are located in sst2 ITIMs (immunoreceptor tyrosine-based inhibitory motifs). This interaction was required for somatostatin-induced SHP-1 recruitment and activation and consequent inhibition of cell proliferation. Src interacted with sst2 and somatostatin promoted a transient Gβγ-dependent Src activation concomitant with sst2 tyrosine hyperphosphorylation and SHP-2 activation. These steps were abrogated with catalytically inactive Src. Both catalytically inactive Src and SHP-2 mutants abolished somatostatin-induced SHP-1 activation and cell growth inhibition. Sst2–Src–SHP-2 complex formation was dynamic. Somatostatin further induced sst2 tyrosine dephosphorylation and complex dissociation accompanied by Src and SHP-2 inhibition. These steps were defective in cells expressing a catalytically inactive Src mutant. All these data suggest that Src acts upstream of SHP-2 in sst2 signaling and provide evidence for a functional role for Src and SHP-2 downstream of an inhibitory G protein-coupled receptor.

The tumor bulk is composed of a highly heterogeneous population of cancer cells, as well as a large variety of resident and infiltrating host cells, extracellular matrix proteins, and secreted proteins, collectively known as the tumor microenvironment (TME). The TME is essential for driving tumor development by promoting cancer cell survival, migration, metastasis, chemoresistance, and the ability to evade the immune system responses. Therapeutically targeting tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), regulatory T-cells (T-regs), and mesenchymal stromal/stem cells (MSCs) is likely to have an impact in cancer treatment. In this review, we focus on describing the normal physiological functions of each of these cell types and their behavior in the cancer setting. Relying on the specific surface markers and secreted molecules in this context, we review the potential targeting of these cells inducing their depletion, reprogramming, or differentiation, or inhibiting their pro-tumor functions or recruitment. Different approaches were developed for this targeting, namely, immunotherapies, vaccines, small interfering RNA, or small molecules.