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The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.

Dermal white adipose tissue (dWAT) is involved in the maintenance of skin homeostasis. However, the studies concerning its molecular regulation are limited. In the present paper, we ask whether the introduction of two transcription factors, Foxn1 and Hif-1α, into the post-wounded skin of Foxn1 mice regulates dWAT during wound healing (days 3 and 6). We have chosen lentivirus vectors (LVs) as a tool to deliver Foxn1 and Hif-1α into the post-wounded skin. We documented that combinations of both transgenes reduces the number, size and diameter of dermal adipocytes at the wound bed area. The qRT-PCR analysis of pro-adipogenic genes, revealed that LV-Hif-1α alone, or combined with LV-Foxn1, increases the mRNA expression of , and at post-wounding day 6. However, the most spectacular stimulatory effect of Foxn1 and/or Hif-1α was observed for Igf2, the growth factor participating in adipogenic signal transduction. Our data also shows that Foxn1/Hif-1α, at post-wounding day 3, reduces levels of and mRNA expression and the percentage of CD68 positive cells in the wound site. In conclusion, the present data are the first to document that Foxn1 and Hif-1α cooperatively (1) regulate dWAT during the proliferative phase of skin wound healing through the Igf2 signaling pathway, and (2) reduce the macrophages content in the wound site.

Current evidence indicates that resistance to the tyrosine kinase-type cell surface receptor (HER2)-targeted therapies is frequently associated with HER3 and active signaling via HER2-HER3 dimers, particularly in the context of breast cancer. Thus, understanding the response to HER2-HER3 signaling and the regulation of the dimer is essential to decipher therapy relapse mechanisms. Here, we investigate a bidirectional relationship between HER2-HER3 signaling and a type-1 transmembrane sorting receptor, sortilin-related receptor (SorLA; SORL1). We demonstrate that heregulin-mediated signaling supports SorLA transcription downstream of the mitogen-activated protein kinase pathway. In addition, we demonstrate that SorLA interacts directly with HER3, forming a trimeric complex with HER2 and HER3 to attenuate lysosomal degradation of the dimer in a Ras-related protein Rab4-dependent manner. In line with a role for SorLA in supporting the stability of the HER2 and HER3 receptors, loss of SorLA compromised heregulin-induced cell proliferation and sensitized metastatic anti-HER2 therapy-resistant breast cancer cells to neratinib in cancer spheroids in vitro and in vivo in a zebrafish brain xenograft model.

Cell migration is the consequence of the sum of positive and negative regulatory mechanisms. Although appropriate migration of neurons is a principal feature of brain development, the negative regulatory mechanisms remain obscure. We found that JNK1 was highly active in developing cortex and that selective inhibition of JNK in the cytoplasm markedly increased both the frequency of exit from the multipolar stage and radial migration rate and ultimately led to an ill-defined cellular organization. Moreover, regulation of multipolar-stage exit and radial migration in Jnk1(-/-) (also known as Mapk8) mice, resulted from consequential changes in phosphorylation of the microtubule regulator SCG10 (also called stathmin-2). Expression of an SCG10 mutant that mimics the JNK1-phosphorylated form restored normal migration in the brains of Jnk1(-/-) mouse embryos. These findings indicate that the phosphorylation of SCG10 by JNK1 is a fundamental mechanism that governs the transition from the multipolar stage and the rate of neuronal cell movement during cortical development.

Background A low tissue oxygen level, < 1% O.sub.2, is a typical characteristic inside of solid tumors in head and neck cancer (HNSCC) affecting a wide array of cell populations, such as macrophages. However, the mechanisms of how hypoxia influences macrophages are not yet fully elucidated. Our research aimed to study the effect of soluble mediators produced by hypoxic cancer cells on macrophage polarization. Furthermore, we studied the effect of a hypoxic microenvironment on the expression of tumorigenic toll-like receptor 9 (TLR9) and the consecutive macrophage polarization. Methods Conditioned media (CM.sub.NOX or CM.sub.HOX) from cell lines UT-SCC-8, UT-SCC-74A, FaDu, MDA-MB-231 and HaCat cultured under normoxic (21% O.sub.2) and hypoxic (1% O.sub.2) conditions were used to polarize human monocyte-derived macrophages. Macrophage polarization was measured by flow cytometry and the production of cytokine mRNA using Taqman qPCR. To study the role of TLR9 in macrophage polarization, the lentiviral CRISPR/Cas9 method was used to establish a stable FaDu.sup.TLR9def clone. Results Our results demonstrate that the soluble mediators produced by the cancer cells under normoxia polarize macrophages towards a hybridized M1/M2a/M2c phenotype. Furthermore, the results suggest that hypoxia has a limited role in altering the array of cancer-produced soluble factors affecting macrophage polarization and cytokine production. Our data also indicates that increased expression of TLR9 due to hypoxia in malignant cells does not markedly influence the polarization of macrophages. TLR9 transcriptional response to hypoxia is dissimilar to a HIF1-[alpha]-regulated LDH-A. This may indicate a context-dependent expression of TLR9 under hypoxia. Conclusions HNSCC cell lines affect both macrophage activity (polarization) and functionality (cytokines), but with exception to iNOS expression, the effects appear independent of hypoxia and TLR9. Keywords: Immune evasion, Anti-cancer immunomodulation, Innate immune response, TLR9, Macrophage polarization, Hypoxia, Immunoediting, Head and neck squamous cell carcinoma

While organ‐confined prostate cancer (PCa) is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumours with concomitant inhibition of two tumour suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having < 50% 5‐year secondary‐therapy‐free patient survival. Functionally, overexpression of PME‐1, a methylesterase for the catalytic PP2A‐C subunit, inhibits anoikis in PTEN‐deficient PCa cells. In vivo, PME‐1 inhibition increased apoptosis in in ovo PCa tumour xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME‐1‐deficient PC3 cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype known to correlate with increased apoptosis sensitivity. In summary, our results demonstrate that PME‐1 supports anoikis resistance in PTEN‐deficient PCa cells. Clinically, these results identify PME‐1 as a candidate biomarker for a subset of particularly aggressive PTEN‐deficient PCa. A subset of prostate cancer (PCa) tumours present simultaneous inactivation of two tumour suppressor phosphatases; phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A). PP2A is inhibited via overexpression of PME‐1. Such cancers are particularly aggressive and often relapse from standard therapy, indicating PME‐1 as a potential clinically applicable biomarker for PCa. Mechanistically, PME‐1 expression protects cancer cells from anoikis, promoting their survival outside the primary tumour.

Background/Aims: Physiological role of luteinizing hormone (LH) and its receptor (LHCGR) in adrenal remains unknown. In inhibin-α/Simian Virus 40 T antigen (SV40Tag) (inhα/Tag) mice, gonadectomy-induced (OVX) elevated LH triggers the growth of transcription factor GATA4 (GATA4)-positive adrenocortical tumors in a hyperplasia-adenoma-adenocarcinoma sequence. Methods: We investigated the role of LHCGR in tumor induction, by crossbreeding inhα/Tag with Lhcgr knockout (LuRKO) mice. By knocking out Lhcgr and Gata4 in Cα1 adrenocortical cells (Lhcgr-ko, Gata4-ko) we tested their role in tumor progression. Results: Adrenal tumors of OVX inhα/Tag mice develop from the hyperplastic cells localized in the topmost layer of zona fasciculata. OVX inhα/Tag/LuRKO only developed SV40Tag positive hyperplastic cells that were GATA4 negative, cleaved caspase-3 positive and did not progress into adenoma. In contrast to Lhcgr-ko, Gata4-ko Cα1 cells presented decreased proliferation, increased apoptosis, decreased expression of Inha, SV40Tag and Lhcgr tumor markers, as well as up-regulated adrenal- and down-regulated sex steroid gene expression. Both Gata4-ko and Lhcgr-ko Cα1 cells had decreased expression of steroidogenic genes resulting in decreased basal progesterone production. Conclusion: Our data indicate that LH/LHCGR signaling is critical for the adrenal cell reprogramming by GATA4 induction prompting adenoma formation and gonadal-like phenotype of the adrenocortical tumors in inhα/Tag mice.

Increased phosphorylation of the KIF5 anterograde motor is associated with impaired axonal transport and neurodegeneration, but paradoxically also with normal transport, though the details are not fully defined. JNK phosphorylates KIF5C on S176 in the motor domain; a site that we show is phosphorylated in brain. Microtubule pelleting assays demonstrate that phosphomimetic KIF5C(1-560)(S176D) associates weakly with microtubules compared to KIF5C(1-560)(WT). Consistent with this, 50% of KIF5C(1-560)(S176D) shows diffuse movement in neurons. However, the remaining 50% remains microtubule bound and displays decreased pausing and increased bidirectional movement. The same directionality switching is observed with KIF5C(1-560)(WT) in the presence of an active JNK chimera, MKK7-JNK. Yet, in cargo trafficking assays where peroxisome cargo is bound, KIF5C(1-560)(S176D)-GFP-FRB transports normally to microtubule plus ends. We also find that JNK increases the ATP hydrolysis of KIF5C in vitro. These data suggest that phosphorylation of KIF5C-S176 primes the motor to either disengage entirely from microtubule tracks as previously observed in response to stress, or to display improved efficiency. The final outcome may depend on cargo load and motor ensembles.

The heat shock (HS) response is crucial for cell survival in harmful environments. Nuclear lamin A/C, encoded by LMNA gene, has been shown to contribute towards altered gene expression during heat shock, but the underlying mechanisms are poorly understood. Here we show that reversible lamin A/C phosphorylation at Ser22 upon HS is an evolutionary conserved stress response that is triggered in concert with HSF1 activation in human and mouse cells and can also be observed in D. melanogaster in vivo. Consequently, the phosphorylation increase facilitated nucleoplasmic localization of lamin A/C and nuclear rounding in response to HS. The importance of lamin phosphorylation equilibria in HS was confirmed by lamin A/C knock-out (KO) cells that showed deformed nuclei after HS and were rescued by ectopic expression of wild-type, but not by a phosphomimetic (S22D) lamin A mutant. Furthermore, HS triggered release of lamina-associated protein 2α (Lap2α) from its association with lamin A/C and concurrently its downregulation, a response that was perturbed in lamin A/C KO cells and in LMNA mutant patient fibroblasts. The abrogated Lap2α response resulted in impaired cell cycle arrest under HS and compromised survival at the recovery. Taken together, our results suggest that the altered phosphorylation stoichiometry of lamin A/C provides an evolutionary conserved mechanism to regulate lamin structure and serve nuclear adaptation and cell survival during HS. Competing Interest Statement The authors have declared no competing interest.

While organ-confined PCa is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumors with concomitantly compromised function of two tumor suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having less than 50% 5-year secondary-therapy free patient survival. Functionally, overexpression of PME-1, a PP2A inhibitor protein, inhibits anoikis in PTEN-deficient PCa cells. In vivo, PME-1 inhibition increased apoptosis in in ovo PCa tumor xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME-1 deficient PCa cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype corresponding to increased apoptosis sensitivity. In summary, we discover that PME-1 overexpression supports anoikis resistance in PTEN-deficient PCa cells. Clinically, the results identify PME-1 as a candidate biomarker for a subset of particularly aggressive PTEN-deficient PCa. Competing Interest Statement The authors have declared no competing interest.