PSMA theragnostics for metastatic castration resistant prostate cancer

There has been tremendous growth in the development of theragnostics for personalized cancer diagnosis and treatment over the past two decades. In prostate cancer, the new generation of prostate specific membrane antigen (PSMA) small molecular inhibitor-based imaging agents achieve extraordinary tumor to background ratios and allow their therapeutic counterparts to deliver effective tumor doses while minimizing normal tissue toxicity. The PSMA targeted small molecule positron emission tomography (PET) agents ¹⁸F-DCFPyL (2-(3-{1-carboxy-5-((6-(18)F-fluoro-pyridine-3-carbonyl)-amino)-pentyl}-ureido)-pentanedioic acid) and Gallium-68 (⁶⁸Ga)-PSMA-11 have been approved by the United States Food and Drug Administration (FDA) for newly diagnosed high risk prostate cancer patients and for patients with biochemical recurrence. More recently, the Phase III VISION trial showed that Lutetium-177 (¹⁷⁷Lu)-PSMA-617 treatment increases progression-free survival and overall survival in patients with heavily pre-treated advanced PSMA-positive metastatic castration-resistant prostate cancer (mCRPC). Here, we review the PSMA targeted theragnostic pairs under clinical investigation for detection and treatment of metastatic prostate cancer.     

Radiothérapie interne vectorisée par 177Lu-PSMA-617 de l’adénocarcinome prostatique métastatique résistant à la castration : à propos d’un cas et revue de la littérature

The prostate-specific membrane antigen (PSMA) is a type II glycoprotein which is over-expressed in prostate cancer tissue, especially in high grade tumours, metastatic disease, as an effect of androgen-deprivation therapies and in castrate-resistant prostate cancer (CRPC). Recent studies about radioligand therapy using PSMA ligands labeled with lutetium-177 (¹⁷⁷Lu) in CRPC patients have suggested its interest as a third line of treatment after second generation anti-androgens and chemotherapy by taxane. We present the case of a CRPC patient who was treated by iterative radioligand therapy using PSMA-617 ligand labeled with ¹⁷⁷Lu. This case illustrates on the one hand the efficacy of the treatment, and on the other hand the fragility of the patients who are at an advanced stage of their disease. Then we present a short review of the literature on this topic, focusing on the published efficacy and tolerance of ¹⁷⁷Lu-PSMA radioligand therapy of CRPC patients.     

The Rapalogue,CCI-779, Improves Salivary Gland Function following Radiation

The standard of care for head and neck cancer typically includes surgical resection of the tumor followed by targeted head and neck radiation. However depending on tumor location and stage, some cases may not require surgical resection while others may be treated with chemoradiation. Unfortunately, these radiation treatments cause chronic negative side effects for patients. These side effects are associated with damage to surrounding normal salivary gland tissue and include xerostomia, changes in taste and malnutrition. The underlying mechanisms of chronic radiation-induced salivary gland dysfunction are unknown, however, in rodent models persistently elevated proliferation is correlated with reduced stimulated salivary flow. The rapalogue, CCI-779, has been used in other cell systems to induce autophagy and reduce proliferation, therefore the aim of this study was to determine if CCI-779 could be utilized to ameliorate chronic radiation-induced salivary gland dysfunction. Four to six week old Atg5^f/f; Aqp5-Cre, Atg5^+/+; Aqp5-Cre and FVB mice were treated with targeted head and neck radiation. FVB mice were treated with CCI-779, chloroquine, or DMSO post-radiation. Stimulated salivary flow rates were determined and parotid and submandibular salivary gland tissues were collected for analyses. Mice with a defect in autophagy, via a conditional knockout of Atg5 in the salivary glands, display increased compensatory proliferation in the acinar cell compartment and hypertrophy at 24-72 hours following radiation. FVB mice treated with post-therapy CCI-779 have significant improvements in salivary gland physiology as determined by stimulated salivary flow rates, proliferation indices and amylase production and secretion. Consequently, post-radiation use of CCI-779 allows for improvement of salivary gland function and reestablishment of glandular homeostasis. As CCI-779 is already FDA approved for other uses, it could have a secondary use to alleviate the chronic side effects in head and neck cancer patients who have completed anti-tumor therapy.     

Aberrant Activation of the RANK Signaling Receptor Induces Murine Salivary Gland Tumors

Unlike cancers of related exocrine tissues such as the mammary and prostate gland, diagnosis and treatment of aggressive salivary gland malignancies have not markedly advanced in decades. Effective clinical management of malignant salivary gland cancers is undercut by our limited knowledge concerning the key molecular signals that underpin the etiopathogenesis of this rare and heterogeneous head and neck cancer. Without knowledge of the critical signals that drive salivary gland tumorigenesis, tumor vulnerabilities cannot be exploited that allow for targeted molecular therapies. This knowledge insufficiency is further exacerbated by a paucity of preclinical mouse models (as compared to other cancer fields) with which to both study salivary gland pathobiology and test novel intervention strategies. Using a mouse transgenic approach, we demonstrate that deregulation of the Receptor Activator of NFkB Ligand (RANKL)/RANK signaling axis results in rapid tumor development in all three major salivary glands. In line with its established role in other exocrine gland cancers (i.e., breast cancer), the RANKL/RANK signaling axis elicits an aggressive salivary gland tumor phenotype both at the histologic and molecular level. Despite the ability of this cytokine signaling axis to drive advanced stage disease within a short latency period, early blockade of RANKL/RANK signaling markedly attenuates the development of malignant salivary gland neoplasms. Together, our findings have uncovered a tumorigenic role for RANKL/RANK in the salivary gland and suggest that targeting this pathway may represent a novel therapeutic intervention approach in the prevention and/or treatment of this understudied head and neck cancer.     

Sox9+ cells are required for salivary gland regeneration after radiation damage via the Wnt/β-catenin pathway

Radiotherapy for head and neck cancer can cause serious side effects, including severe damage to the salivary glands, resulting in symptoms such as xerostomia, dental caries, and oral infection. Because of the lack of long-term treatment for the symptoms of xerostomia, current research has focused on finding endogenous stem cells that can differentiate into various cell lineages to replace lost tissues and restore functions. Here, we report that Sox9⁺ cells can differentiate into various salivary epithelial cell lineages under homeostatic conditions. After ablating Sox9⁺ cells, the salivary glands of irradiated mice showed more severe phenotypes and the reduced proliferative capacity. Analysis of online single-cell RNA-sequencing data reveals the enrichment of the Wnt/β-catenin pathway in the Sox9⁺ cell population. Furthermore, treatment with a Wnt/β-catenin inhibitor in irradiated mice inhibits the regenerative capability of Sox9⁺ cells. Finally, we show that Sox9⁺ cells are capable of forming organoids in vitro and that transplanting these organoids into salivary glands after radiation partially restored salivary gland functions. These results suggest that regenerative therapy targeting Sox9⁺ cells is a promising approach to treat radiation-induced salivary gland injury.     

Early and Late Toxicity of Radioiodine Therapy: Detection and Management

ObjectiveTo review the early and late toxicity associated with radioiodine (¹³¹I) therapy, highlighting the need for early detection and, where possible, preventive measures.MethodsWe performed a literature search on MEDLINE using the terms radioiodine, ¹³¹I toxicity, complications, Graves disease, and thyroid cancer and chose the most relevant studies for this review. Where appropriate, we refer to our own published series of patients as well.ResultsUptake of ¹³¹I into the salivary glands, lacrimal glands, fetal thyroid, and adult thyroid accounts for the early toxic effects of radioiodine therapy. Delayed radiation effects to the gonads, bone marrow, and cell nuclei give rise to late complications. Toxicity may also arise from uptake into metastatic tumors located at vulnerable sites, including the spinal cord, brain, and lungs.ConclusionAlthough radioiodine therapy for benign and malignant thyroid disorders is usually well tolerated, clinicians involved in the management of thyroid disorders need to be aware of the potential toxicity of radioiodine and take all measures to reduce these effects to a minimum. (Endocr Pract. 2010;16:1064-1070)     

Prevention of Radiation-Induced Salivary Gland Dysfunction Utilizing a CDK Inhibitor in a Mouse Model

Background

Treatment of head and neck cancer with radiation often results in damage to surrounding normal tissues such as salivary glands. Permanent loss of function in the salivary glands often leads patients to discontinue treatment due to incapacitating side effects. It has previously been shown that IGF-1 suppresses radiation-induced apoptosis and enhances G2/M arrest leading to preservation of salivary gland function. In an effort to recapitulate the effects of IGF-1, as well as increase the likelihood of translating these findings to the clinic, the small molecule therapeutic Roscovitine, is being tested. Roscovitine is a cyclin-dependent kinase inhibitor that acts to transiently inhibit cell cycle progression and allow for DNA repair in damaged tissues.

Methodology/Principal Findings

Treatment with Roscovitine prior to irradiation induced a significant increase in the percentage of cells in the G₂/M phase, as demonstrated by flow cytometry. In contrast, mice treated with radiation exhibit no differences in the percentage of cells in G₂/M when compared to unirradiated controls. Similar to previous studies utilizing IGF-1, pretreatment with Roscovitine leads to a significant up-regulation of p21 expression and a significant decrease in the number of PCNA positive cells. Radiation treatment leads to a significant increase in activated caspase-3 positive salivary acinar cells, which is suppressed by pretreatment with Roscovitine. Administration of Roscovitine prior to targeted head and neck irradiation preserves normal tissue function in mouse parotid salivary glands, both acutely and chronically, as measured by salivary output.

Conclusions/Significance

These studies suggest that induction of transient G₂/M cell cycle arrest by Roscovitine allows for suppression of apoptosis, thus preserving normal salivary function following targeted head and neck irradiation. This could have an important clinical impact by preventing the negative side effects of radiation therapy in surrounding normal tissues.     

Marrow-Derived Autologous Stromal Cells for the Restoration of Salivary Hypofunction (MARSH): Study protocol for a phase 1 dose-escalation trial of patients with xerostomia after radiation therapy for head and neck cancer: MARSH: Marrow-Derived Autologous Stromal Cells for the Restoration of Salivary Hypofunction

BackgroundXerostomia, or dry mouth, is a common side effect of head and neck radiation. Current treatment options for radiation-induced xerostomia are generally supportive in nature. Adult stem cells are the ultimate source for replenishment of salivary gland tissue. Bone marrow–derived mesenchymal stromal cells (BM-MSCs) are a viable cell-based therapy for xerostomia. We have undertaken studies enabling U.S. Food and Drug Administration Investigational New Drug status, demonstrating the normal phenotype, intact functionality, and pro-growth secretome of interferon-γ (IFNγ)-stimulated BM-MSCs taken from patients with head and neck cancer who have undergone radiation ± chemotherapy. Here we present the protocol of MARSH, a first-in-human clinical trial of bone marrow–derived, IFNγ-activated BM-MSCs for the treatment of radiation-induced xerostomia.MethodsThis single-center phase 1 dose-escalation with expansion cohort, non–placebo-controlled study will assess the safety and tolerability of BM-MSCs for the treatment of radiation-induced xerostomia in patients who had head and neck cancer. The phase 1 dose-escalation study will be a 3 + 3 design with staggered enrollment. A total of 21 to 30 subjects (9 to 18 in phase 1 study, 12 in expansion cohort) will be enrolled. The primary endpoint is determining the recommended phase 2 dose (RP2D) of IFNγ-stimulated BM-MSCs to enable further studies on the efficacy of BM-MSCs. Patients’ bone marrow will be aspirated, and BM-MSCs will be expanded, stimulated with IFNγ, and injected into the submandibular gland. The RP2D will be determined by dose-limiting toxicities occurring within 1 month of BM-MSC injection. Secondary outcomes of saliva amounts and composition, ultrasound of salivary glands, and quality of life surveys will be taken at 3-, 6-, 12-, and 24-month visits.DiscussionAutotransplantation of IFNγ-stimulated BM-MSCs in salivary glands after radiation therapy or chemoradiation therapy may provide an innovative remedy to treat xerostomia and restore quality of life. This is the first therapy for radiation-induced xerostomia that may be curative.Trial registrationWorld Health Organization International Clinical Trials Registry Platform: NCT04489732     

Suppression of Radiation-Induced Salivary Gland Dysfunction by IGF-1

Background

Radiation is a primary or secondary therapeutic modality for treatment of head and neck cancer. A common side effect of irradiation to the neck and neck region is xerostomia caused by salivary gland dysfunction. Approximately 40,000 new cases of xerostomia result from radiation treatment in the United States each year. The ensuing salivary gland hypofunction results in significant morbidity and diminishes the effectiveness of anti-cancer therapies as well as the quality of life for these patients. Previous studies in a rat model have shown no correlation between induction of apoptosis in the salivary gland and either the immediate or chronic decrease in salivary function following γ-radiation treatment.

Methodology/Principal Finding

A significant level of apoptosis can be detected in the salivary glands of FVB mice following γ-radiation treatment of the head and neck and this apoptosis is suppressed in transgenic mice expressing an activated mutant of Akt (myr-Akt1). Importantly, this suppression of apoptosis in myr-Akt1 mice preserves salivary function, as measured by saliva output, three and thirty days after γ-radiation treatment. In order to translate these studies into a preclinal model we found that intravenous injection of IGF1 stimulated activation of endogenous Akt in the salivary glands in vivo. A single injection of IGF1 prior to exposure to γ-radiation diminishes salivary acinar cell apoptosis and completely preserves salivary gland function three and thirty days following irradiation.

Conclusions/Significance

These studies suggest that apoptosis of salivary acinar cells underlies salivary gland hypofunction occurring secondary to radiation of the head and neck region. Targeted delivery of IGF1 to the salivary gland of patients receiving head and neck irradiation may be useful in reducing or eliminating xerostomia and restoring quality of life to these patients.     

Calcium signaling defects underlying salivary gland dysfunction

Salivary glands secrete saliva, a mixture of proteins and fluids, which plays an extremely important role in the maintenance of oral health. Loss of salivary secretion causes a dry mouth condition, xerostomia, which has numerous deleterious consequences including opportunistic infections within the oral cavity, difficulties in eating and swallowing food, and problems with speech. Saliva secretion is regulated by stimulation of specific signaling mechanisms within the acinar cells of the gland. Neurotransmitter-stimulated increase in cytosolic [Ca²⁺] ([Ca²⁺]_i) in acinar cells is the primary trigger for salivary fluid secretion from salivary glands, the loss of which is a critical factor underlying dry mouth conditions in patients. The increase in [Ca²⁺]_i regulates multiple ion channel and transport activities that together generate the osmotic gradient which drives fluid secretion across the apical membrane. Ca²⁺ entry mediated by the Store-Operated Ca²⁺ Entry (SOCE) mechanism provides the essential [Ca²⁺]_i signals to trigger salivary gland fluid secretion. Under physiological conditions depletion of ER-Ca²⁺ stores is caused by activation of IP3R by IP3 and this provides the stimulus for SOCE. Core components of SOCE in salivary gland acinar cells are the plasma membrane Ca²⁺ channels, Orai1 and TRPC1, and STIM1, a Ca²⁺-sensor protein in the ER, which regulates both channels. In addition, STIM2 likely enhances the sensitivity of cells to ER-Ca²⁺ depletion thereby tuning the cellular response to agonist stimulation. Two major, clinically relevant, conditions which cause irreversible salivary gland dysfunction are radiation treatment for head-and-neck cancers and the autoimmune exocrinopathy, Sjögren's syndrome (pSS). However, the exact mechanism(s) that causes the loss of fluid secretion, in either condition, is not clearly understood. A number of recent studies have identified that defects in critical Ca²⁺ signaling mechanisms underlie salivary gland dysfunction caused by radiation treatment or Sjögren's syndrome (pSS). This chapter will discuss these very interesting and important studies.     

Simplified patient-specific renal dosimetry in 177Lu therapy: a proof of concept

PurposeThe aim of this proof-of-concept study is to propose a simplified personalized kidney dosimetry procedure in ¹⁷⁷Lu peptide receptor radionuclide therapy (PRRT) for neuroendocrine tumors and metastatic prostate cancer. It relies on a single quantitative SPECT/CT acquisition and multiple radiometric measurements executed with a collimated external probe, properly directed on kidneys.MethodsWe conducted a phantom study involving external count-rate measurements in an abdominal phantom setup filled with activity concentrations of ^99mTc, reproducing patient-relevant organ effective half-lives occurring in ¹⁷⁷Lu PRRT. GATE Monte Carlo (MC) simulations of the experiment, using ^99mTc and ¹⁷⁷Lu as sources, were performed. Furthermore, we tested this method via MC on a clinical case of ¹⁷⁷Lu-DOTATATE PRRT with SPECT/CT images at three time points (2, 20 and 70 hrs), comparing a simplified kidney dosimetry, employing a single SPECT/CT and probe measurements at three time points, with the complete MC dosimetry.ResultsThe experimentally estimated kidney half-life with background subtraction applied was compatible within 3% with the expected value. The MC simulations of the phantom study, both with ^99mTc and ¹⁷⁷Lu, confirmed a similar level of accuracy. Concerning the clinical case, the simplified dosimetric method led to a kidney dose estimation compatible with the complete MC dosimetry within 6%, 12% and 2%, using respectively the SPECT/CT at 2, 20 and 70 hrs.ConclusionsThe proposed simplified procedure provided a satisfactory accuracy and would reduce the imaging required to derive the kidney absorbed dose to a unique quantitative SPECT/CT, with consequent benefits in terms of clinic workflows and patient comfort.     

Androgens as therapy for androgen receptor-positive castration-resistant prostate cancer

Prostate cancer is the most frequently diagnosed non-cutaneous tumor of men in Western countries. While surgery is often successful for organ-confined prostate cancer, androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. Shortening the period of androgen ablation therapy may benefit prostate cancer patients. Intermittent Androgen Deprivation therapy improves quality of life, reduces toxicity and medical costs, and delays disease progression in some patients. Cell culture and xenograft studies using androgen receptor (AR)-positive castration-resistant human prostate cancers cells (LNCaP, ARCaP, and PC-3 cells over-expressing AR) suggest that androgens may suppress the growth of AR-rich prostate cancer cells. Androgens cause growth inhibition and G1 cell cycle arrest in these cells by regulating c-Myc, Skp2, and p27^Kip via AR. Higher dosages of testosterone cause greater growth inhibition of relapsed tumors. Manipulating androgen/AR signaling may therefore be a potential therapy for AR-positive advanced prostate cancer.     

骨髓来源细胞治疗放射性唾液腺损伤的研究进展

罹患头颈部肿瘤的患者在接受放射治疗时往往会发生放射性唾液腺损伤。射线的照射使患者唾液腺结构破坏、功能减退,患者的生活质量严重下降。对于放射性唾液腺损伤,临床上尚无有效的治疗方式。骨髓来源细胞(bone marrow-derived cells,BMDCs)最早用于治疗血液系统疾病。随着对BMDCs认识的逐渐深入,BMDCs的应用领域日益广泛。近些年来,一些动物实验的研究结果表明,利用BMDCs治疗放射性唾液腺损伤能够有效地保护腺体内各种实质细胞,促进腺组织再生,恢复唾液腺功能。本文主要对利用BMDCs治疗放射性唾液腺损伤的治疗方式、治疗效果及其主要的治疗机制进行综述,并对该领域今后的研究方向进行了展望。     

Imaging prostate derived tumors with PET and N-(3-[18F]fluoropropyl)putrescine

Because of the high uptake of polyamines by the prostate and by prostate derived tumors, polyamines have been considered as potential imaging agents for metastatic prostate cancer. We now report the successful PET imaging of the Dunning R3327H prostatic carcinoma with N-(3-[¹⁸F]fluoropropyl)-putrescine (FPP), a positron-labeled putrescine analog. Additionally, the biodistribution of FPP in tumor bearing Copenhagen male rats is analyzed. The tumor uptake of FPP was high, and the tumor-to-muscle ratios at 1,2,3 and 4.5 h post-injection were 7.2 ± 1.0, 5.61 ± 1.65, 4.62 ± 0.21 and 3.51 ± 0.91 respectively. The estimated radiation dose for FPP was calculated from rat biodistribution data. The radiation dose estimates suggest that the critical organ, following the administration of FPP, is the upper large intenstine which receives 0.3 rad/mCi administered.     

Radioprotective Effect of Lidocaine on Neurotransmitter Agonist-Induced Secretion in Irradiated Salivary Glands

Background

Previously we verified the radioprotective effect of lidocaine on the function and ultrastructure of salivary glands in rabbits. However, the underlying mechanism of lidocaine''s radioprotective effect is unknown. We hypothesized that lidocaine, as a membrane stabilization agent, has a protective effect on intracellular neuroreceptor-mediated signaling and hence can help preserve the secretory function of salivary glands during radiotherapy.

Methods and Materials

Rabbits were irradiated with or without pretreatment with lidocaine before receiving fractionated radiation to a total dose of 35 Gy. Sialoscintigraphy and saliva total protein assay were performed before radiation and 1 week after the last radiation fraction. Isolated salivary gland acini were stimulated with either carbachol or adrenaline. Ca²⁺ influx in response to the stimulation with these agonists was measured using laser scanning confocal microscopy.

Results

The uptake of activity and the excretion fraction of the parotid glands were significantly reduced after radiation, but lidocaine had a protective effect. Saliva total protein concentration was not altered after radiation. For isolated acini, Ca²⁺ influx in response to stimulation with carbachol, but not adrenaline, was impaired after irradiation; lidocaine pretreatment attenuated this effect.

Conclusions

Lidocaine has a radioprotective effect on the capacity of muscarinic agonist-induced water secretion in irradiated salivary glands.     

SPM Receptor Expression and Localization in Irradiated Salivary Glands

Radiation therapy–mediated salivary gland destruction is characterized by increased inflammatory cell infiltration and fibrosis, both of which ultimately lead to salivary gland hypofunction. However, current treatments (e.g., artificial saliva and sialagogues) only promote temporary relief of symptoms. As such, developing alternative measures against radiation damage is critical for restoring salivary gland structure and function. One promising option for managing radiation therapy–mediated damage in salivary glands is by activation of specialized proresolving lipid mediator receptors due to their demonstrated role in resolution of inflammation and fibrosis in many tissues. Nonetheless, little is known about the presence and function of these receptors in healthy and/or irradiated salivary glands. Therefore, the goal of this study was to detect whether these specialized proresolving lipid mediator receptors are expressed in healthy salivary glands and, if so, if they are maintained after radiation therapy–mediated damage. Our results indicate that specialized proresolving lipid mediator receptors are heterogeneously expressed in inflammatory as well as in acinar and ductal cells within human submandibular glands and that their expression persists after radiation therapy. These findings suggest that epithelial cells as well as resident immune cells represent potential targets for modulation of resolution of inflammation and fibrosis in irradiated salivary glands.     

Clinical experience with gene therapy for the treatment of prostate cancer

Localized prostate cancer can be treated effectively with radical prostatectomy or radiation therapy. The treatment options for metastatic prostate cancer are limited to hormonal therapy; hormone-refractory cancer is treated with taxane-based chemotherapy, which provides only a modest survival benefit. New treatments are needed. The gene for the initiation of prostate cancer has not been identified; however, gene therapy can involve tumor injection of a gene to kill cells, systemic gene delivery to target and kill metastases, or local gene expression intended to generate a systemic response. This review will provide an overview of the various strategies of cancer gene therapy, focusing on those that have gone to clinical trial, detailing clinical experience in prostate cancer patients.     

Expression of gastric gland mucous cell-type mucin in normal and neoplastic human tissues.

Gastric gland mucous cells produce class III mucin, which is also found in Brunner's glands and mucous glands along the pancreaticobiliary tract, and in metaplasia and adenocarcinomas differentiating towards gastric mucosa. Recently, we showed that class III mucin possesses GlcNAcalpha1-->4Galbeta-->R, formed by alpha1,4-N-acetylglucosaminyltransferase (alpha4GnT). Examining the tissue-specific expression of mucin epitopes is useful to clarify cell-lineage differentiation and to identify the site of origin of metastatic carcinomas in histological specimens. Formalin-fixed, paraffin-embedded tissue sections from esophagus, stomach, colon, liver, pancreas, lung, kidney, prostate, breast, and salivary gland resected for carcinoma, as well as salivary gland adenoma, colon adenoma, and metastatic adenocarcinoma of lymph nodes from stomach, pancreas, colon, and breast, were immunostained for MUC6, alpha4GnT, and GlcNAcalpha1-->4Galbeta-->R. These were all expressed in normal, metaplastic, and adenocarcinoma tissues of stomach, pancreas, and bile duct, and in pulmonary mucinous bronchioloalveolar carcinomas. Cells expressing alpha4GnT uniformly expressed GlcNAcalpha1-->4Galbeta-->R. Only MUC6 was expressed in normal salivary glands, pancreas, seminal vesicles, renal tubules, and colon adenomas, and in normal tissue and adenocarcinomas of prostate and breast. No tissues showed immunoreactivity for alpha4GnT alone. Immunohistochemistry (IHC) profiles were similar for metastatic carcinomas and primary carcinoma tissues. The IHC profiles for MUC6, alpha4GnT, and GlcNAcalpha1-->4Galbeta-->R may be diagnostically relevant.     

The engulfment receptor Draper is required for autophagy during cell death

Autophagy is a process to degrade and recycle cytoplasmic contents. Autophagy is required for survival in response to starvation, but has also been associated with cell death. How autophagy functions during cell survival in some contexts and cell death in others is unknown. Drosophila larval salivary glands undergo programmed cell death requiring autophagy genes, and are cleared in the absence of known phagocytosis. Recently, we demonstrated that Draper (Drpr), the Drosophila homolog of C. elegans engulfment receptor CED-1, is required for autophagy induction during cell death, but not during cell survival. drpr mutants fail to clear salivary glands. drpr knockdown in salivary glands prevents the induction of autophagy, and Atg1 misexpression in drpr null mutants suppresses salivary gland persistence. Surprisingly, drpr knockdown cell-autonomously prevents autophagy induction in dying salivary gland cells, but not in larval fat body cells following starvation. This is the first engulfment factor shown to function in cellular self-clearance, and the first report of a cell-death-specific autophagy regulator.Key words: autophagy, Draper, programmed cell death, engulfment, developmentProgrammed cell death is required for animal development and tissue homeostasis. Improper cell death leads to pathologies including autoimmunity and cancer. Several morphological forms of cell death occur during animal development, including apoptosis and autophagic cell death. Autophagic cell death is characterized by the presence of autophagosomes in dying cells that are not known to be engulfed by phagocytes. Autophagic cell death is observed during several types of mammalian developmental cell death, including regression of the corpus luteum and involution of mammary and prostate glands.During macroautophagy (autophagy), cytoplasmic components are sequestered by autophagosomes and delivered to the lysosome for degradation. Autophagy is a cellular response to stress required for survival in response to starvation. Whereas autophagy has been associated with cell death, it is unknown how autophagy is distinguished during cell death and cell survival. Autophagy is induced in Drosophila in response to starvation in the fat body where it promotes cell survival, while autophagy is induced by the steroid hormone ecdysone in salivary glands where it promotes cell death. This allows studies of autophagy in different cell types and in response to different stimuli.Drosophila larval salivary glands die with autophagic cell death morphology and autophagy is required for their degradation. Expression of the caspase inhibitor p35 enhances salivary gland persistence in Atg mutants, suggesting that caspases and autophagy function in parallel during salivary gland degradation. Either activation of caspases or Atg1 misexpression is sufficient to induce ectopic salivary gland clearance. We queried genome-wide microarray data from purified dying salivary glands and noted the induction of engulfment genes, those required for a phagocyte to consume and degrade a dying cell. We also noted few detectable changes in engulfment genes in Drosophila larvae during starvation.We found that Drpr, the Drosophila orthologue of C. elegans engulfment receptor CED-1, is enriched in dying salivary glands, and drpr null mutants have persistent salivary glands. Interestingly, whereas knockdown of drpr in phagocytic blood cells fails to influence salivary gland clearance, expression of drpr-RNAi in salivary glands prevents gland clearance. Drosophila drpr is alternatively spliced to produce three isoforms. We found that drpr-I-specific knockdown prevents salivary gland degradation and Drpr-I expression in salivary glands of drpr null mutants rescues salivary gland persistence. Therefore, drpr is autonomously required for salivary gland clearance. However, how Drpr is induced or activated during hormone-regulated cell death remains to be determined.drpr knockdown fails to influence caspase activation, and caspase inhibitor p35 expression in drpr null mutants enhances salivary gland persistence, suggesting that Drpr functions downstream or parallel to caspases in dying salivary glands. Interestingly, we found that drpr knockdown in salivary glands prevents the formation of GFP-LC3 puncta. Further, Atg1 misexpression in salivary glands of drpr null mutants suppresses salivary gland persistence. drpr is therefore required for autophagy induction in salivary glands, and Atg1 functions downstream of Drpr in this tissue. We found that several other engulfment genes are required for salivary gland degradation. However, the Drpr signaling mechanism leading to autophagy induction in salivary glands remains to be elucidated.We tested whether drpr is a general regulator of autophagy. The Drosophila fat body is a nutrient storage and mobilization organ akin to the mammalian liver, and is a well-established model to study starvation-induced autophagy. We found that drpr-RNAi expression in fat body clone cells fails to prevent GFP-Atg8 puncta formation in response to starvation. Similarly, drpr null fat body clone cells form Cherry-Atg8 puncta after starvation. Strikingly, drpr-RNAi expression in salivary gland clone cells inhibits the formation of GFP-Atg8 puncta. Therefore, drpr is cell-autonomously required for autophagy induction in dying salivary gland cells, but not for autophagy induction in fat body cells after starvation. These findings suggest that distinct signaling mechanisms regulate autophagy in response to nutrient deprivation compared to steroid hormone induction. Little is known about what distinguishes autophagy function in cell survival versus death. It is possible that varying levels of autophagy are induced during specific cell contexts and that high levels of autophagy could overwhelm a cell—leading to cell death. Autophagic degradation of specific cargo, such as cell death inhibitors, could also contribute to cell death.Given recent interest in manipulation of autophagy for therapies, it is possible that factors such as Drpr could be used as biomarkers to distinguish autophagy leading to cell death versus cell survival. While it is generally accepted that augmentation of protein clearance by autophagy during neurodegeneration would be beneficial, the role of autophagy in tumor progression is less clear. For example, monoallelic loss of the human Atg6 homolog beclin 1 is prevalent in human cancers, suggesting that autophagy is a tumorsuppressive mechanism. Thus, autophagy enhancers have been proposed for cancer prevention. However, autophagy occurs in tumor cells as a survival mechanism, and autophagy inhibitors have been proposed for anti-cancer therapies. Understanding how autophagy is regulated in different contexts is critical for appropriate therapeutic strategies.