An Antibody-Drug Conjugate Directed to Tissue Factor Shows Preclinical Antitumor Activity in Head and Neck Cancer as a Single Agent and in Combination with Chemoradiotherapy.

Head and neck squamous cell carcinoma (HNSCC) is a solid tumor type that arises in the squamous epithelial cells lining the mucosal surfaces of the upper aerodigestive tract. Long-term survival of patients with advanced disease stage remains disappointing with current treatment options. We show that tissue factor is abundantly expressed on patient-derived HNSCC cell lines, xenograft tumor material, and tumor biopsies from patients with HNSCC. Tisotumab vedotin (TV) is an antibody-drug conjugate (ADC) directed to tissue factor, a protein expressed in many solid tumors. HNSCC cells and xenograft tumors were efficiently eliminated in vitro and in vivo with TV-monotherapy compared with treatment with a control antibody conjugated to monomethyl auristatin E (MMAE). Antitumor activity of TV was also tested in vivo in combination with chemoradiotherapy, standard of care for patients with advanced stage HNSCC tumors outside the oral cavity. Preclinical studies showed that by adding TV to chemoradiotherapy, survival was markedly improved, and TV, not radiotherapy or chemotherapy, was the main driver of antitumor activity. Interestingly, TV-induced cell death in xenograft tumors showed an influx of macrophages indicative of a potential immune-mediated mode-of-action. In conclusion, on the basis of these preclinical data, TV may be a novel treatment modality for patients suffering from head and neck cancer and is hypothesized to improve efficacy of chemoradiotherapy. SIGNIFICANCE: This work shows preclinical in vitro and in vivo antitumor activity of the antibody-drug conjugate Tisotumab vedotin in head and neck cancer models, and enhanced activity in combination with chemoradiotherapy, supporting further clinical development for this cancer type.

Mechanistic and pharmacodynamic studies of DuoBody-CD3x5T4 in preclinical tumor models.

CD3 bispecific antibodies (bsAbs) show great promise as anticancer therapeutics. Here, we show in-depth mechanistic studies of a CD3 bsAb in solid cancer, using DuoBody-CD3x5T4. Cross-linking T cells with tumor cells expressing the oncofetal antigen 5T4 was required to induce cytotoxicity. Naive and memory CD4+ and CD8+ T cells were equally effective at mediating cytotoxicity, and DuoBody-CD3x5T4 induced partial differentiation of naive T-cell subsets into memory-like cells. Tumor cell kill was associated with T-cell activation, proliferation, and production of cytokines, granzyme B, and perforin. Genetic knockout of FAS or IFNGR1 in 5T4+ tumor cells abrogated tumor cell kill. In the presence of 5T4+ tumor cells, bystander kill of 5T4- but not of 5T4-IFNGR1- tumor cells was observed. In humanized xenograft models, DuoBody-CD3x5T4 antitumor activity was associated with intratumoral and peripheral blood T-cell activation. Lastly, in dissociated patient-derived tumor samples, DuoBody-CD3x5T4 activated tumor-infiltrating lymphocytes and induced tumor-cell cytotoxicity, even when most tumor-infiltrating lymphocytes expressed PD-1. These data provide an in-depth view on the mechanism of action of a CD3 bsAb in preclinical models of solid cancer.

Preclinical Characterization and Phase I Trial Results of a Bispecific Antibody Targeting PD-L1 and 4-1BB (GEN1046) in Patients with Advanced Refractory Solid Tumors.

Checkpoint inhibitors (CPI) have revolutionized the treatment paradigm for advanced solid tumors; however, there remains an opportunity to improve response rates and outcomes. In preclinical models, 4-1BB costimulation synergizes with CPIs targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis by activating cytotoxic T-cell-mediated antitumor immunity. DuoBody-PD-L1×4-1BB (GEN1046) is an investigational, first-in-class bispecific immunotherapy agent designed to act on both pathways by combining simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation in one molecule. GEN1046 induced T-cell proliferation, cytokine production, and antigen-specific T-cell-mediated cytotoxicity superior to clinically approved PD-(L)1 antibodies in human T-cell cultures and exerted potent antitumor activity in transplantable mouse tumor models. In dose escalation of the ongoing first-in-human study in heavily pretreated patients with advanced refractory solid tumors (NCT03917381), GEN1046 demonstrated pharmacodynamic immune effects in peripheral blood consistent with its mechanism of action, manageable safety, and early clinical activity [disease control rate: 65.6% (40/61)], including patients resistant to prior PD-(L)1 immunotherapy. SIGNIFICANCE: DuoBody-PD-L1×4-1BB (GEN1046) is a first-in-class bispecific immunotherapy with a manageable safety profile and encouraging preclinical and early clinical activity. With its ability to confer clinical benefit in tumors typically less sensitive to CPIs, GEN1046 may fill a clinical gap in CPI-relapsed or refractory disease or as a combination therapy with CPIs. See related commentary by Li et al., p. 1184. This article is highlighted in the In This Issue feature, p. 1171.

IGF2 is a potential factor in RAI-refractory differentiated thyroid cancer.

Differentiated thyroid cancer (DTC) is the most frequent endocrine tumor with a good prognosis after primary treatment in most cases. By contrast, 30-40% of patients with metastatic DTC are unresponsive to 131I radioactive iodide (RAI) treatment due to tumor dedifferentiation. Currently, underlying molecular mechanisms of dedifferentiation remain elusive and predictive biomarkers are lacking. Therefore, the present study aimed to identify molecular biomarkers in primary tumors associated with RAI refractoriness. A retrospective cohort was gathered consisting of RAI-sensitive patients with DTC and RAI-refractory patients with poorly DTC. In all patients, extensive intratumoral mutation profiling, gene fusions analysis, telomerase reverse transcriptase (TERT) promoter mutation analysis and formalin-fixed paraffin-embedded-compatible RNA sequencing were performed. Genetic analyses revealed an increased mutational load in RAI-refractory DTC, including mutations in AKT1, PTEN, TP53 and TERT promoter. Transcriptomic analyses revealed profound differential expression of insulin-like growth factor 2 (IGF2), with up to 100-fold higher expression in RAI-refractory DTC compared with in RAI-sensitive DTC cases. ELISA revealed significant lower IGF2 plasma concentrations after surgery and subsequent 131I RAI therapy in patients with DTC compared with pretreatment baseline. Overall, the current findings suggested that the tumor-promoting growth factor IGF2 may have a potential role in acquiring RAI refractoriness.

Cooperative Targeting of Immunotherapy-Resistant Melanoma and Lung Cancer by an AXL-Targeting Antibody-Drug Conjugate and Immune Checkpoint Blockade.

Although immune checkpoint blockade (ICB) has shown remarkable clinical benefit in a subset of patients with melanoma and lung cancer, most patients experience no durable benefit. The receptor tyrosine kinase AXL is commonly implicated in therapy resistance and may serve as a marker for therapy-refractory tumors, for example in melanoma, as we previously demonstrated. Here, we show that enapotamab vedotin (EnaV), an antibody-drug conjugate targeting AXL, effectively targets tumors that display insensitivity to immunotherapy or tumor-specific T cells in several melanoma and lung cancer models. In addition to its direct tumor cell killing activity, EnaV treatment induced an inflammatory response and immunogenic cell death in tumor cells and promoted the induction of a memory-like phenotype in cytotoxic T cells. Combining EnaV with tumor-specific T cells proved superior to either treatment alone in models of melanoma and lung cancer and induced ICB benefit in models otherwise insensitive to anti-PD-1 treatment. Our findings indicate that targeting AXL-expressing, immunotherapy-resistant tumors with EnaV causes an immune-stimulating tumor microenvironment and enhances sensitivity to ICB, warranting further investigation of this treatment combination. SIGNIFICANCE: These findings show that targeting AXL-positive tumor fractions with an antibody-drug conjugate enhances antitumor immunity in several humanized tumor models of melanoma and lung cancer.

Digoxin treatment reactivates in vivo radioactive iodide uptake and correlates with favorable clinical outcome in non-medullary thyroid cancer.

PURPOSE: Non-medullary thyroid cancer (NMTC) treatment is based on the ability of thyroid follicular cells to accumulate radioactive iodide (RAI). However, in a subset of NMTC patients tumor dedifferentiation occurs, leading to RAI resistance. Digoxin has been demonstrated to restore iodide uptake capacity in vitro in poorly differentiated and anaplastic NMTC cells, termed redifferentiation. The aim of the present study was to investigate the in vivo effects of digoxin in TPO-Cre/LSL-BrafV600E mice and digoxin-treated NMTC patients. METHODS: Mice with thyroid cancer were subjected to 3D ultrasound for monitoring tumor growth and 124I PET/CT for measurement of intratumoral iodide uptake. Post-mortem analyses on tumor tissues comprised gene expression profiling and measurement of intratumoral autophagy activity. Through PALGA (Dutch Pathology Registry), archived tumor material was obtained from 11 non-anaplastic NMTC patients who were using digoxin. Clinical characteristics and tumor material of these patients were compared to 11 matched control NMTC patients never treated with digoxin. RESULTS: We found that in mice, tumor growth was inhibited and 124I accumulation was sustainably increased after short-course digoxin treatment. Post-mortem analyses revealed that digoxin treatment increased autophagy activity and enhanced expression of thyroid-specific genes in mouse tumors compared to vehicle-treated mice. Digoxin-treated NMTC patients exhibited significantly higher autophagy activity and a higher differentiation status as compared to matched control NMTC patients, and were associated with favourable clinical outcome. CONCLUSIONS: These in vivo data support the hypothesis that digoxin may represent a repositioned adjunctive treatment modality that suppresses tumor growth and improves RAI sensitivity in patients with RAI-refractory NMTC.

Akt1 genetic variants confer increased susceptibility to thyroid cancer.

The PI3K-Akt-mTOR pathway plays a central role in the development of non-medullary thyroid carcinoma (NMTC). Although somatic mutations have been identified in these genes in NMTC patients, the role of germline variants has not been investigated. Here, we selected frequently occurring genetic variants in AKT1, AKT2, AKT3, PIK3CA and MTOR and have assessed their effect on NMTC susceptibility, progression and clinical outcome in a Dutch discovery cohort (154 patients, 188 controls) and a Romanian validation cohort (159 patients, 260 controls). Significant associations with NMTC susceptibility were observed for AKT1 polymorphisms rs3803304, rs2494732 and rs2498804 in the Dutch discovery cohort, of which the AKT1 rs3803304 association was confirmed in the Romanian validation cohort. No associations were observed between PI3K-Akt-mTOR polymorphisms and clinical parameters including histology, TNM staging, treatment response and clinical outcome. Functionally, cells bearing the associated AKT1 rs3803304 risk allele exhibit increased levels of phosphorylated Akt protein, potentially leading to elevated signaling activity of the oncogenic Akt pathway. All together, germline encoded polymorphisms in the PI3K-Akt-mTOR pathway could represent important risk factors in development of NMTC.

GWAS of thyroid stimulating hormone highlights pleiotropic effects and inverse association with thyroid cancer.

Thyroid stimulating hormone (TSH) is critical for normal development and metabolism. To better understand the genetic contribution to TSH levels, we conduct a GWAS meta-analysis at 22.4 million genetic markers in up to 119,715 individuals and identify 74 genome-wide significant loci for TSH, of which 28 are previously unreported. Functional experiments show that the thyroglobulin protein-altering variants P118L and G67S impact thyroglobulin secretion. Phenome-wide association analysis in the UK Biobank demonstrates the pleiotropic effects of TSH-associated variants and a polygenic score for higher TSH levels is associated with a reduced risk of thyroid cancer in the UK Biobank and three other independent studies. Two-sample Mendelian randomization using TSH index variants as instrumental variables suggests a protective effect of higher TSH levels (indicating lower thyroid function) on risk of thyroid cancer and goiter. Our findings highlight the pleiotropic effects of TSH-associated variants on thyroid function and growth of malignant and benign thyroid tumors.

Assessing thyroid cancer risk using polygenic risk scores.

Genome-wide association studies (GWASs) have identified at least 10 single-nucleotide polymorphisms (SNPs) associated with papillary thyroid cancer (PTC) risk. Most of these SNPs are common variants with small to moderate effect sizes. Here we assessed the combined genetic effects of these variants on PTC risk by using summarized GWAS results to build polygenic risk score (PRS) models in three PTC study groups from Ohio (1,544 patients and 1,593 controls), Iceland (723 patients and 129,556 controls), and the United Kingdom (534 patients and 407,945 controls). A PRS based on the 10 established PTC SNPs showed a stronger predictive power compared with the clinical factors model, with a minimum increase of area under the receiver-operating curve of 5.4 percentage points (P ≤ 1.0 × 10-9). Adding an extended PRS based on 592,475 common variants did not significantly improve the prediction power compared with the 10-SNP model, suggesting that most of the remaining undiscovered genetic risk in thyroid cancer is due to rare, moderate- to high-penetrance variants rather than to common low-penetrance variants. Based on the 10-SNP PRS, individuals in the top decile group of PRSs have a close to sevenfold greater risk (95% CI, 5.4-8.8) compared with the bottom decile group. In conclusion, PRSs based on a small number of common germline variants emphasize the importance of heritable low-penetrance markers in PTC.

Decreased Aerobic Exercise Capacity After Long-Term Remission From Cushing Syndrome: Exploration of Mechanisms.

BACKGROUND: Although major improvements are achieved after cure of Cushing syndrome (CS), fatigue and decreased quality of life persist. This is the first study to measure aerobic exercise capacity in patients in remission of CS for more than 4 years in comparison with matched controls, and to investigate whether the reduction in exercise capacity is related to alterations in muscle tissue. METHODS: Seventeen patients were included. A control individual, matched for sex, estrogen status, age, body mass index, smoking, ethnicity, and physical activity level was recruited for each patient. Maximal aerobic capacity (VO2peak) was assessed during incremental bicycle exercise to exhaustion. In 8 individually matched patients and controls, a percutaneous muscle biopsy was obtained and measures were made of cross-sectional areas, capillarization, and oxphos complex IV (COXIV) protein content as an indicator of mitochondrial content. Furthermore, protein content of endothelial nitric oxide synthase (eNOS) and eNOS phosphorylated on serine1177 and of the NAD(P)H-oxidase subunits NOX2, p47phox, and p67phox were measured in the microvascular endothelial layer. FINDINGS: Patients showed a lower mean VO2peak (SD) (28.0 [7.0] vs 34.8 [7.9] ml O2/kg bw/min, P < .01), maximal workload (SD) (176 [49] vs 212 [67] watt, P = .01), and oxygen pulse (SD) (12.0 [3.7] vs 14.8 [4.2] ml/beat, P < .01) at VO2peak. No differences were seen in muscle fiber type-specific cross-sectional area, capillarization measures, mitochondrial content, and protein content of eNOS, eNOS-P-ser1177, NOX2, p47phox, and p67phox. INTERPRETATION: Because differences in muscle fiber and microvascular outcome measures are not statistically significant, we hypothesize that cardiac dysfunction, seen in active CS, persists during remission and limits blood supply to muscles.

Exploring the Role of IL-32 in HIV-Related Kaposi Sarcoma.

The intracellular proinflammatory mediator IL-32 is associated with tumor progression; however, the mechanisms remain unknown. We studied IL-32 mRNA expression as well as expression of other proinflammatory cytokines and mediators, including IL-1α, IL-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α, the proangiogenic and antiapoptotic enzyme cyclooxygenase-2, the IL-8 receptor C-X-C chemokine receptor (CXCR) 1, and the intracellular kinase focal adhesion kinase-1. The interaction of IL-32 expression with expression of IL-6, TNF-α, IL-8, and cyclooxygenase-2 was also investigated. Biopsy specimens of 11 HIV-related, 7 non-HIV-related Kaposi sarcoma (KS), and 7 normal skin tissues (NSTs) of Dutch origin were analyzed. RNA was isolated from the paraffin material, and gene expression levels of IL-32 α, ß, and γ isoforms, IL1a, IL1b, IL6, IL8, TNFA, PTGS2, CXCR1, and PTK2 were determined using real-time quantitative PCR. Significantly higher expression of IL-32ß and IL-32γ isoforms was observed in HIV-related KS biopsy specimens compared with non-HIV-related KS and NST. The splicing ratio of the IL-32 isoforms showed IL-32γ as the highest expressed isoform, followed by IL-32ß, in HIV-related KS cases compared with non-HIV-related KS and NST. Our data suggest a possible survival mechanism by the splicing of IL-32γ to IL-32ß and also IL-6, IL-8, and CXCR1 signaling pathways to reverse the proapoptotic effect of the IL-32γ isoform, leading to tumor cell survival and thus favoring tumor progression.

Digitalislike Compounds Restore hNIS Expression and Iodide Uptake Capacity in Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is a rare malignancy that accounts for 1%-2% of all thyroid cancers. ATC is one of the most aggressive human cancers, with rapid growth, tumor invasion, and development of distant metastases. The median survival is only 5 mo, and the 1-y survival is less than 20%. Moreover, as a result of severe dedifferentiation, including the loss of human sodium iodide symporter (hNIS) expression, radioactive iodide (RAI) therapy is ineffective. Recently, we have demonstrated beneficial effects of autophagy-activating digitalislike compounds (DLCs) on redifferentiation and concomitant restoration of iodide uptake in RAI-refractory papillary and follicular thyroid cancer cell lines. In the current study, the effects of DLCs on differentiation and proliferation of ATC cell lines were investigated. Methods: Autophagy activity was assessed in ATC patient tissues by immunofluorescent staining for the autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3). In addition, the effect of autophagy-activating DLCs on the proliferation, gene expression profile, and iodide uptake capacity of ATC cell lines was studied. Results: Diminished autophagy activity was observed in ATC tissues, and in vitro treatment of ATC cell lines with DLCs robustly restored hNIS and thyroglobulin expression and iodide uptake capacity. In addition, proliferation was strongly reduced by induction of cell cycle arrest and, to some extent, cell death. Mechanistically, reactivation of functional hNIS expression could be attributed to activation of the transcription factors activating transcription factor 3 and protooncogene c-fosConclusion: DLCs could represent a promising adjunctive therapy for restoring iodide avidity within the full spectrum from RAI-refractory dedifferentiated to ATC.

Pathological processes and therapeutic advances in radioiodide refractory thyroid cancer.

While in most patients with non-medullary thyroid cancer (TC), disease remission is achieved by thyroidectomy and ablation of tumor remnants by radioactive iodide (RAI), a substantial subgroup of patients with metastatic disease present tumor lesions that have acquired RAI resistance as a result of dedifferentiation. Although oncogenic mutations in BRAF, TERT promoter and TP53 are associated with an increased propensity for induction of dedifferentiation, the role of genetic and epigenetic aberrations and their effects on important intracellular signaling pathways is not yet fully elucidated. Also immune, metabolic, stemness and microRNA pathways have emerged as important determinants of TC dedifferentiation and RAI resistance. These signaling pathways have major clinical implications since their targeting could inhibit TC progression and could enable redifferentiation to restore RAI sensitivity. In this review, we discuss the current insights into the pathological processes conferring dedifferentiation and RAI resistance in TC and elaborate on novel advances in diagnostics and therapy to improve the clinical outcome of RAI-refractory TC patients.

Increased Adipocyte Size, Macrophage Infiltration, and Adverse Local Adipokine Profile in Perirenal Fat in Cushing's Syndrome.

OBJECTIVE: To analyze changes in fat cell size, macrophage infiltration, and local adipose tissue adipokine profiles in different fat depots in patients with active Cushing's syndrome. METHODS: Subcutaneous (SC) and perirenal (PR) adipose tissue of 10 patients with Cushing's syndrome was compared to adipose tissue of 10 gender-, age-, and BMI-matched controls with regard to adipocyte size determined by digital image analysis on hematoxylin and eosin stainings, macrophage infiltration determined by digital image analysis on CD68 stainings, and adipose tissue leptin and adiponectin levels using fluorescent bead immunoassays and ELISA techniques. RESULTS: Compared to the controls, mean adipocyte size was larger in PR adipose tissue in patients. The percentage of macrophage infiltration of the PR adipose tissue and PR adipose tissue lysate leptin levels were higher and adiponectin levels were lower in SC and PR adipose tissue lysates in patients. The adiponectin levels were also lower in the SC adipose tissue supernatants of patients. Associations were found between the severity of hypercortisolism and PR adipocyte size. CONCLUSIONS: Cushing's syndrome is associated with hypertrophy of PR adipocytes and a higher percentage of macrophage infiltration in PR adipose tissue. These changes are associated with an adverse local adipokine profile.

Association of NF-κB polymorphisms with clinical outcome of non-medullary thyroid carcinoma.

The NF-κB inflammatory pathway plays a major role in cancer development and clinical progression. Activation of NF-κB signaling is promoted by NFKB1 and inhibited by NFKBIA. The present study aimed to determine the relevance of NFKB1 rs4648068 and NFKBIA rs2233406 genetic variants for non-medullary thyroid cancer (NMTC) susceptibility, progression and clinical outcome. This case-control and cohort study consists of a Romanian discovery cohort (157 patients and 258 controls) and a Dutch validation cohort (138 patients and 188 controls). In addition, patient cohorts were analyzed further for the association of genetic variants with clinical parameters. Functional studies were performed on human peripheral blood mononuclear cells. No associations were observed between the studied genetic variants and TC susceptibility. Although no statistically significant associations with clinical parameters were observed for NFKB1 rs4648068, the heterozygous genotype of NFKBIA rs2233406 was correlated with decreased radioactive iodide sensitivity requiring higher cumulative dosages to achieve clinical response. These findings were discovered in the Romanian cohort (P < 0.001) and confirmed in the Dutch cohort (P = 0.01). Functional studies revealed that this NFKBIA rs2233406 genotype was associated with elevated TLR4-mediated IL-1ß production. In conclusion, genetic variation in NFKBIA, an inhibitor of NF-κB signaling, is associated with clinical response to RAI therapy and with increased production of the pro-inflammatory cytokine IL-1ß, providing a potential mechanism for the observed clinical associations. These data suggest that NF-κB signaling is involved in NMTC pathogenesis and that the inflammatory tumor microenvironment could contribute to RAI resistance.

A genome-wide association study yields five novel thyroid cancer risk loci.

The great majority of thyroid cancers are of the non-medullary type. Here we report findings from a genome-wide association study of non-medullary thyroid cancer, including in total 3,001 patients and 287,550 controls from five study groups of European descent. Our results yield five novel loci (all with Pcombined<3 × 10-8): 1q42.2 (rs12129938 in PCNXL2), 3q26.2 (rs6793295 a missense mutation in LRCC34 near TERC), 5q22.1 (rs73227498 between NREP and EPB41L4A), 10q24.33 (rs7902587 near OBFC1), and two independently associated variants at 15q22.33 (rs2289261 and rs56062135; both in SMAD3). We also confirm recently published association results from a Chinese study of a variant on 5p15.33 (rs2736100 near the TERT gene) and present a stronger association result for a moderately correlated variant (rs10069690; OR=1.20, P=3.2 × 10-7) based on our study of individuals of European ancestry. In combination, these results raise several opportunities for future studies of the pathogenesis of thyroid cancer.

Digitalis-like Compounds Facilitate Non-Medullary Thyroid Cancer Redifferentiation through Intracellular Ca2+, FOS, and Autophagy-Dependent Pathways.

Up to 20%-30% of patients with metastatic non-medullary thyroid cancer have persistent or recurrent disease resulting from tumor dedifferentiation. Tumor redifferentiation to restore sensitivity to radioactive iodide (RAI) therapy is considered a promising strategy to overcome RAI resistance. Autophagy has emerged as an important mechanism in cancer dedifferentiation. Here, we demonstrate the therapeutic potential of autophagy activators for redifferentiation of thyroid cancer cell lines. Five autophagy-activating compounds, all known as digitalis-like compounds, restored hNIS expression and iodide uptake in thyroid cancer cell lines. Upregulation of hNIS was mediated by intracellular Ca2+ and FOS activation. Cell proliferation was inhibited by downregulating AKT1 and by induction of autophagy and p21-dependent cell-cycle arrest. Digitalis-like compounds, also designated as cardiac glycosides for their well-characterized beneficial effects in the treatment of heart disease, could therefore represent a promising repositioned treatment modality for patients with RAI-refractory thyroid carcinoma. Mol Cancer Ther; 16(1); 169-81. ©2016 AACR.

Autophagy activity is associated with membranous sodium iodide symporter expression and clinical response to radioiodine therapy in non-medullary thyroid cancer.

Although non-medullary thyroid cancer (NMTC) generally has a good prognosis, 30-40% of patients with distant metastases develop resistance to radioactive iodine (RAI) therapy due to tumor dedifferentiation. For these patients, treatment options are limited and prognosis is poor. In the present study, expression and activity of autophagy was assessed in large sets of normal, benign and malignant tissues and was correlated with pathology, SLC5A5/hNIS (solute carrier family 5 member 5) protein expression, and with clinical response to RAI ablation therapy in NMTC patients. Fluorescent immunostaining for the autophagy marker LC3 was performed on 100 benign and 80 malignant thyroid tissues. Semiquantitative scoring was generated for both diffuse LC3-I intensity and number of LC3-II-positive puncta and was correlated with SLC5A5 protein expression and clinical parameters. Degree of diffuse LC3-I intensity and number of LC3-II-positive puncta scoring were not discriminative for benign vs. malignant thyroid lesions. Interestingly, however, in NMTC patients significant associations were observed between diffuse LC3-I intensity and LC3-II-positive puncta scoring on the one hand and clinical response to RAI therapy on the other hand (odds ratio [OR] = 3.13, 95% confidence interval [CI] =1.91-5.12, P = 0.01; OR = 5.68, 95%CI = 3.02-10.05, P = 0.002, respectively). Mechanistically, the number of LC3-II-positive puncta correlated with membranous SLC5A5 expression (OR = 7.71, 95%CI = 4.15-11.75, P<0.001), number of RAI treatments required to reach remission (P = 0.014), cumulative RAI dose (P = 0.026) and with overall remission and recurrence rates (P = 0.031). In conclusion, autophagy activity strongly correlates with clinical response of NMTC patients to RAI therapy, potentially by its capacity to maintain tumor cell differentiation and to preserve functional iodide uptake.

Alternatively spliced isoforms of IL-32 differentially influence cell death pathways in cancer cell lines.

Alternative splicing is a biological mechanism that enables the synthesis of several isoforms with different or even opposite functions. This process must be tightly regulated to prevent unwanted isoform expression favoring pathological processes. Some isoforms of interleukin 32 (IL-32) are reported to be more potent in inducing inflammation, however the role in cell death remains to be investigated. This study demonstrates that IL-32γ and IL-32ß can induce caspase-8-dependent cell death whereas this was not observed for IL-32α. Overexpression of IL-32ß or IL-32γ but not IL-32α, resulted in enhanced expression of the survival cytokine IL-8. Furthermore, restoring the IL-8 signaling pathway by overexpressing CXCR1 in HEK293 cells, rescued IL-32ß but not IL-32γ-induced cell death. Interestingly, IL-32γ was able to downregulate CXCR1 and thereby induce cell death. Subsequent studies into the role of IL-32 in thyroid cancer (TC) revealed that several IL-32 isoforms, IL-8, and CXCR1 are expressed in TC cell lines and specimens. Remarkably, TC cell lines were found to produce high concentrations of IL-8, indicating an important role for IL-8 in the survival-signaling pathway in these cells. Intriguingly, a significant correlation between the IL-8 receptor CXCR1 and IL-32γ was observed in TC specimens, while this was not observed for the other IL-32 splice variants. Blocking IL-32 alternative splicing by Isoginkgetin resulted in predominant expression of IL-32γ splice variants and cell death in TC cell lines. All together, modulation of IL-32 alternative splicing could represent a novel strategy for the treatment of malignancies, in particular thyroid cancer.