Patients with head and neck squamous cell carcinoma (HNSCC) who actively smoke during treatment have worse survival compared with never-smokers and former-smokers. We hypothesize the poor prognosis ...in tobacco smokers with HNSCC is, at least in part, due to ongoing suppression of immune response. We characterized the tumor immune microenvironment (TIM) of HNSCC in a retrospective cohort of 177 current, former, and never smokers.
Tumor specimens were subjected to analysis of CD3, CD8, FOXP3, PD-1, PD-L1, and pancytokeratin by multiplex immunofluorescence, whole-exome sequencing, and RNA sequencing. Immune markers were measured in tumor core, tumor margin, and stroma.
Our data indicate that current smokers have significantly lower numbers of CD8
cytotoxic T cells and PD-L1
cells in the TIM compared with never- and former-smokers. While tumor mutation burden and mutant allele tumor heterogeneity score do not associate with smoking status, gene-set enrichment analyses reveal significant suppression of IFNα and IFNγ response pathways in current smokers. Gene expression of canonical IFN response chemokines,
,
, and
, are lower in current smokers than in former smokers, suggesting a mechanism for the decreased immune cell migration to tumor sites.
These results suggest active tobacco use in HNSCC has an immunosuppressive effect through inhibition of tumor infiltration of cytotoxic T cells, likely as a result of suppression of IFN response pathways. Our study highlights the importance of understanding the interaction between smoking and TIM in light of emerging immune modulators for cancer management.
Neurotrophins and their receptors are frequently expressed in malignant gliomas, yet their functions are largely unknown. Previously, we have shown that p75 neurotrophin receptor is required for ...glioma invasion and proliferation. However, the role of Trk receptors has not been examined. In this study, we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here, we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC, not TrkA, and they also express neurotrophins NGF, BDNF, and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely, TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further, pharmacological inhibition of both ERK and Akt pathways blocked BDNF, and NT3 stimulated BTIC survival. Importantly, attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling, and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma.
Background: The role of Trk neurotrophin receptors in glioma is unknown.
Results: TrkB and TrkC are required for survival of brain tumor-initiating cells in the absence of EGF and FGF.
Conclusion: Trk receptors can control the survival of BTICs in the absence of EGF and FGF.
Significance: Trks may be important targets for treatment of malignant gliomas.
Immune infiltration is typically quantified using cellular density, not accounting for cellular clustering. Tumor-associated macrophages (TAM) activate oncogenic signaling through paracrine ...interactions with tumor cells, which may be better reflected by local cellular clustering than global density metrics. Using multiplex immunohistochemistry and digital pathologic analysis we quantified cellular density and cellular clustering for myeloid cell markers in 129 regions of interest from 55 samples from 35 patients with metastatic ccRCC. CD68+ cells were found to be clustered with tumor cells and dispersed from stromal cells, while CD163+ and CD206+ cells were found to be clustered with stromal cells and dispersed from tumor cells. CD68+ density was not associated with OS, while high tumor/CD68+ cell clustering was associated with significantly worse OS. These novel findings would not have been identified if immune infiltrate was assessed using cellular density alone, highlighting the importance of including spatial analysis in studies of immune cell infiltration of tumors. Significance: Increased clustering of CD68+ TAMs and tumor cells was associated with worse overall survival for patients with metastatic ccRCC. This effect would not have been identified if immune infiltrate was assessed using cell density alone, highlighting the importance of including spatial analysis in studies of immune cell infiltration of tumors.
Estrogens play an important role in the development of breast cancer. Inhibiting 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1)—the enzyme responsible for the last step in the biosynthesis of the ...most potent estrogen, estradiol (E
2)—would thus allow hindering the growth of estrogen-sensitive tumors. Based on a previous study identifying 16β-benzyl-E
2 (
1) as a lead compound for developing inhibitors of the transformation of estrone (E
1) into E
2, we modified the benzyl group of
1 to improve its inhibitory activity. Three strategies were also devised to produce compounds with less residual estrogenic activity: (1) replacing the hydroxy group by a hydrogen at position 3 (C3); (2) adding a methoxy at C2; and (3) adding an alkylamide chain known to be antiestrogenic at C7. In order to test the inhibitory potency of the new compounds, we used the human breast cancer cell line T-47D, which exerts a strong endogenous 17β-HSD1 activity. In this intact cell model, 16β-
m-carbamoylbenzyl-E
2 (
4m) emerged as a potent inhibitor of 17β-HSD1 with an IC
50 value of 44
nM for the transformation of
14C-E
1 (60
nM) into
14C-E
2 (24-h incubation). In another assay aimed at assessing the unwanted estrogenic activity, a 10-day treatment with
4m at a concentration of 0.5
μM induced some proliferation (38%) of T-47D estrogen-sensitive (ER
+) breast cancer cells. Interestingly, when
4m (0.5
μM) was given with E
1 (0.1
nM) in a 10-day treatment, it blocked 62% of the T-47D cell proliferation induced by E
1 after its reduction to E
2 by 17β-HSD1. Thus, in addition to generating useful structure–activity relationships for the development of 17β-HSD1 inhibitors, our study demonstrates that using such inhibitors is a valuable strategy for reducing the level of E
2 and consequently its proliferative effect in T-47D ER
+ breast cancer cells.
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17Beta-hydroxysteroid dehydrogenase type 3 (17β-HSD3) is a steroidogenic enzyme that catalyzes the transformation of 4-androstene-3,17-dione (Δ
4-dione) into androgen testosterone ...(T). To provide effective inhibitors of androgen biosynthesis, we synthesized two different series (amines and carbamates) of 3β-substituted-androsterone derivatives and we tested their inhibitory activity on 17β-HSD3. From the results of our structure–activity relationship study, we identified a series of compounds producing a strong inhibition of 17β-HSD3 overexpressed in HEK-293 cells (homogenized cells). The most active compound when tested in intact HEK-293 transfected cells, namely (3α,5α)-3-{
trans-2,5-dimethyl-4-{2-(trifluoromethyl)phenyl sulfonyl}piperazin-1-ylmethyl}-3-hydroxyandrostan-17-one (
15b), shows an IC
50 value of 6
nM, this compound is thus eight times more active than our reference compound D-5-2 (IC
50
=
51
nM). This new improved inhibitor did not stimulate the proliferation of androgen-sensitive Shionogi cells, suggesting a non-androgenic profile. Compound
15b is thus a good candidate for further in vivo studies on rodents.
•Lactone- and lactol-estradiol derivatives were synthesized and characterized.•Lactone E-ring was diversified by adding a hydroxymethyl, a methylcarboxylate, a carboxy or an allyl group.•A chemical ...approach was developed to introduce a chemical group on hindered beta-steroid face.•Lactone and lactol derivatives inhibited 17β-HSD1 (34–60%) similarly as the natural substrate estrone (53%).
To control estradiol (E2) formation, we are interested in synthesizing inhibitors of 17β-hydroxyteroid dehydrogenase type 1 (17β-HSD1). Since the results of docking experiments have shown that E2-lactone derivatives substituted in position 19 or 20 (E-ring) could generate interactions with the active site of the enzyme, we carried out their chemical synthesis. After having prepared the 16β,17β-γ-lactone-E2 in four steps starting from estrone (E1), we introduced the molecular diversity by adding a hydroxymethyl, a methylcarboxylate, a carboxy or an allyl group. The allyl derivative was used as a key intermediate to generate a hydroxyethyl side chain in α or β position. Two lactols were also obtained from two hydroxyalkyl lactones. Enzymatic assays revealed that lactone and lactol derivatives weakly inhibited 17β-HSD1 in homogenized HEK-293 cells overexpressing 17β-HSD1 (34–60% at 1μM) and in intact T-47D cells expressing 17β-HSD1 (10–40% at 10μM).
This article is part of a Special Issue entitled “Synthesis and biological testing of steroid derivatives as inhibitors”.
The involvement of aromatase, steroid sulfatase (STS) and reductive 17β-hydroxysteroid dehydrogenases (17β-HSDs) in the production of estrogens was determined in four cell lines of endometrial cancer ...(Ishikawa, HEC-1A, HEC-1B and RL-95) and one cell line of cervix cancer (Hela) in culture. After incubation with 4-androstene-3,17-dione (4-dione), there are no estrogens, estrone (E1) and estradiol (E2), detected suggesting that the pathway of aromatase is not important in these cell lines. In whole cells, the results show low percentages of transformation of estrone sulfate (E1S) into E1 suggesting that the entrance of E1S is difficult. However, in homogenized cells the STS activity was much higher and fully blocked by an inhibitor. Using selective inhibitors for each reductive 17β-HSD (types 1, 5, 7 and 12), alone or in combination, we did not succeed in completely blocking the conversion of E1 into E2, suggesting that another 17β-HSD (known or unknown) is involved in the formation of E2 from E1.
•17β-HSD3 is involved in the formation of androgenic hormone testosterone (T).•RM-532-105 is an androsterone derivative inhibiting 17β-HSD3.•RM-532-105 inhibits the formation of T in homogenized and ...whole HEK-293 cells overexpressing 17β-HSD3.•RM-532-105 (10mg/kg, s.c.) reached a plasma concentration of 250ng/mL at 7h in rat.
17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3 or HSD17B3) catalyzes the last step in the biosynthesis of the potent androgen testosterone (T), by stereoselectively reducing the C17 ketone of 4-androstene-3,17-dione (4-dione), with NADPH as cofactor. Since T plays an important role in androgen-sensitive diseases, this enzyme is thus an interesting therapeutic target. In an attempt to design compounds to lower the level of T, we synthesized androsterone derivatives substituted at position 3 as inhibitors of 17β-HSD3, and selected one of the most potent compounds for additional studies. In an enzymatic assay in homogenized and whole HEK-293 cells overexpressing 17β-HSD3, the inhibitor RM-532-105 efficiently inhibited the conversion of natural substrate 4-dione (50nM) into T with an IC50 of 26nM and 5nM, respectively. Moreover, the inhibitor RM-532-105 (10mg/kg) reached a plasma concentration of 250ng/mL at 7h (AUC 24h: 3485ngh/mL) after subcutaneous (s.c.) injection in the rat. In order to mimic the human situation in which 4-dione is converted to T in the testis, we used intact rats. Treatment for 7 days with 17β-HSD3 inhibitor RM-532-105 by s.c. injection or oral gavage exerted no effect on the testis, prostate and seminal vesicle weight and no modification in the levels of plasma steroids. However, after this treatment, the concentration of inhibitor in plasma increased depending on the dose. We thereafter determined the concentration of inhibitor in the testis and we discovered that the compound was slightly present. In fact, at 10mg/kg, the inhibitor RM-532-105 seems to have difficulty penetrating inside the testis and was found to be concentrated in the testicular capsule, and therefore unable to inhibit the 17β-HSD3 located inside the testis. However, with a higher dose of 50mg/kg injected s.c. in rats, RM-532-105 significantly decreased the level of T and dihydrotestosterone measured in plasma at 2h.
Abstract
Introduction
We investigated a commercially available sequencing panel to study the effect of sequencing depth, variant calling strategy, and targeted sequencing region on identifying ...tumor‐derived variants in cell‐free bronchoalveolar lavage (cfBAL) DNA compared with plasma cfDNA.
Methods
Sequencing was performed at low or high coverage using two filtering algorithms to identify tumor variants on two panels targeting 77 and 197 genes respectively.
Results
One hundred and four sequencing files from 40 matched DNA samples of cfBAL, plasma, germline leukocytes, and archival tumor specimens in 10 patients with early‐stage lung cancer were analyzed. By low‐coverage sequencing, tumor‐derived cfBAL variants were detected in 5/10 patients (50%) compared with 2/10 (20%) for plasma. High‐coverage sequencing did not affect the number of tumor‐derived variants detected in either biospecimen type. Accounting for germline mutations eliminated false‐positive plasma calls regardless of coverage (0/10 patients with tumor‐derived variants identified) and increased the number of cfBAL calls (5/10 patients with tumor‐derived variants identified). These results were not affected by the number of targeted genes.
•17β-HSD3 is involved in the formation of androgenic hormone testosterone (T).•RM-532-105 is an androsterone derivative inhibiting 17β-HSD3.•RM-532-105 inhibits the formation of T in homogenized and ...whole HEK-293 cells overexpressing 17β-HSD3.•RM-532-105 (10mg/kg, s.c.) reached a plasma concentration of 250ng/mL at 7h in rat.
17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3 or HSD17B3) catalyzes the last step in the biosynthesis of the potent androgen testosterone (T), by stereoselectively reducing the C17 ketone of 4-androstene-3,17-dione (4-dione), with NADPH as cofactor. Since T plays an important role in androgen-sensitive diseases, this enzyme is thus an interesting therapeutic target. In an attempt to design compounds to lower the level of T, we synthesized androsterone derivatives substituted at position 3 as inhibitors of 17β-HSD3, and selected one of the most potent compounds for additional studies. In an enzymatic assay in homogenized and whole HEK-293 cells overexpressing 17β-HSD3, the inhibitor RM-532-105 efficiently inhibited the conversion of natural substrate 4-dione (50nM) into T with an IC50 of 26nM and 5nM, respectively. Moreover, the inhibitor RM-532-105 (10mg/kg) reached a plasma concentration of 250ng/mL at 7h (AUC 24h: 3485ngh/mL) after subcutaneous (s.c.) injection in the rat. In order to mimic the human situation in which 4-dione is converted to T in the testis, we used intact rats. Treatment for 7 days with 17β-HSD3 inhibitor RM-532-105 by s.c. injection or oral gavage exerted no effect on the testis, prostate and seminal vesicle weight and no modification in the levels of plasma steroids. However, after this treatment, the concentration of inhibitor in plasma increased depending on the dose. We thereafter determined the concentration of inhibitor in the testis and we discovered that the compound was slightly present. In fact, at 10mg/kg, the inhibitor RM-532-105 seems to have difficulty penetrating inside the testis and was found to be concentrated in the testicular capsule, and therefore unable to inhibit the 17β-HSD3 located inside the testis. However, with a higher dose of 50mg/kg injected s.c. in rats, RM-532-105 significantly decreased the level of T and dihydrotestosterone measured in plasma at 2h.
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