EGFR inhibitors exacerbate differentiation and cell cycle arrest induced by retinoic acid and vitamin D3 in acute myeloid leukemia cells

By means of an unbiased, automated fluorescence microscopy-based screen, we identified the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib as potent enhancers of the differentiation of HL-60 acute myeloid leukemia (AML) cells exposed to suboptimal concentrations of vitamin A (all-trans retinoic acid, ATRA) or vitamin D (1α,25-hydroxycholecalciferol, VD). Erlotinib and gefitinib alone did not promote differentiation, yet stimulated the acquisition of morphological and biochemical maturation markers (including the expression of CD11b and CD14 as well as increased NADPH oxidase activity) when combined with either ATRA or VD. Moreover, the combination of erlotinib and ATRA or VD synergistically induced all the processes that are normally linked to terminal hematopoietic differentiation, namely, a delayed proliferation arrest in the G₀/G₁ phase of the cell cycle, cellular senescence, and apoptosis. Erlotinib potently inhibited the (auto)phosphorylation of mitogen-activated protein kinase 14 (MAPK14, best known as p38^MAPK) and SRC family kinases (SFKs). If combined with the administration of ATRA or VD, the inhibition of p38^MAPK or SFKs with specific pharmacological agents mimicked the pro-differentiation activity of erlotinib. These data were obtained with 2 distinct AML cell lines (HL-60 and MOLM-13 cells) and could be confirmed on primary leukemic blasts isolated from the circulation of AML patients. Altogether, these findings point to a new regimen for the treatment of AML, in which naturally occurring pro-differentiation agents (ATRA or VD) may be combined with EGFR inhibitors.     

Molecular mechanisms of ATP secretion during immunogenic cell death

The immunogenic demise of cancer cells can be induced by various chemotherapeutics, such as anthracyclines and oxaliplatin, and provokes an immune response against tumor-associated antigens. Thus, immunogenic cell death (ICD)-inducing antineoplastic agents stimulate a tumor-specific immune response that determines the long-term success of therapy. The release of ATP from dying cells constitutes one of the three major hallmarks of ICD and occurs independently of the two others, namely, the pre-apoptotic exposure of calreticulin on the cell surface and the postmortem release of high-mobility group box 1 (HMBG1) into the extracellular space. Pre-mortem autophagy is known to be required for the ICD-associated secretion of ATP, implying that autophagy-deficient cancer cells fail to elicit therapy-relevant immune responses in vivo. However, the precise molecular mechanisms whereby ATP is actively secreted in the course of ICD remain elusive. Using a combination of pharmacological screens, silencing experiments and techniques to monitor the subcellular localization of ATP, we show here that, in response to ICD inducers, ATP redistributes from lysosomes to autolysosomes and is secreted by a mechanism that requires the lysosomal protein LAMP1, which translocates to the plasma membrane in a strictly caspase-dependent manner. The secretion of ATP additionally involves the caspase-dependent activation of Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)-mediated, myosin II-dependent cellular blebbing, as well as the opening of pannexin 1 (PANX1) channels, which is also triggered by caspases. Of note, although autophagy and LAMP1 fail to influence PANX1 channel opening, PANX1 is required for the ICD-associated translocation of LAMP1 to the plasma membrane. Altogether, these findings suggest that caspase- and PANX1-dependent lysosomal exocytosis has an essential role in ATP release as triggered by immunogenic chemotherapy.     

Premortem autophagy determines the immunogenicity of chemotherapy-induced cancer cell death

One particular strategy to render anticancer therapies efficient consists of converting the patient's own tumor cells into therapeutic vaccines, via the induction of immunogenic cell death (ICD). One of the hallmarks of ICD dwells in the active release of ATP by cells committed to undergo, but not yet having succumbed to, apoptosis. We observed that the knockdown of essential autophagy-related genes (ATG3, ATG5, ATG7 and BECN1) abolishes the pre-apoptotic secretion of ATP by several human and murine cancer cell lines undergoing ICD. Accordingly, autophagy-competent, but not autophagy-deficient, tumor cells treated with ICD inducers in vitro could induce a tumor-specific immune response in vivo. Cancer cell lines stably depleted of ATG5 or ATG7 normally generate tumors in vivo, and such autophagy-deficient neoplasms, upon systemic treatment with ICD inducers, exhibit the same levels of apoptosis (as monitored by nuclear shrinkage and caspase-3 activation) and necrosis (as determined by following the kinetics of HMGB1 release) as their autophagy-proficient counterparts. However, autophagy-incompetent cancers fail to release ATP, to recruit immune effectors into the tumor bed and to respond to chemotherapy in conditions in which autophagy-competent tumors do so. The intratumoral administration of ecto-ATPase inhibitors increases extracellular ATP concentrations, re-establishes the therapy-induced recruitment of dendritic cells and T cells into the tumor bed, and restores the chemotherapeutic response of autophagy-deficient cancers. Altogether, these results suggest that autophagy-incompetent tumor cells escape from chemotherapy-induced (and perhaps natural?) immunosurveillance because they are unable to release ATP.     

The antiestrogen ICI 182,780 induces early effects on the adult male mouse reproductive tract and long-term decreased fertility without testicular atrophy

Background  

Estrogen receptors (ER) have important physiological roles in both the female and male reproductive systems. Previous studies using the estrogen receptor-α knockout mouse (αERKO) or antiestrogen treatment in adult rodents have shown that ERα is essential for normal function of the male reproductive tract. In the present study, time-response effects of the antiestrogen ICI 182,780 were determined to better understand ERα function in the adult male.     

Crosstalk between ER stress and immunogenic cell death

Preclinical and clinical findings suggest that tumor-specific immune responses may be responsible – at least in part – for the clinical success of therapeutic regimens that rely on immunogenic cell death (ICD) inducers, including anthracyclines and oxaliplatin. The molecular pathways whereby some, but not all, cytotoxic agents promote bona fide ICD remain to be fully elucidated. Nevertheless, a central role for the endoplasmic reticulum (ER) stress response has been revealed in all scenarios of ICD described thus far. Hence, components of the ER stress machinery may constitute clinically relevant druggable targets for the induction of ICD. In this review, we will summarize recent findings in the field of ICD research with a special focus on ER stress mechanisms and their implication for cancer therapy.