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Head and neck squamous cell carcinoma represents a complex set of neoplasms arising in diverse anatomical locations. The site and stage of the cancer determine whether patients will be treated with single or multi-modality therapy. The HDAC inhibitor LBH589 is effective in treating some haematological neoplasms and shows promise for certain epithelial neoplasms. As with other human cancer cell lines, LBH589 causes up-regulation of p21, G2/M cell cycle arrest, and cell death of human HNSCC cell lines, as measured using flow cytometry and cDNA microarrays. Global RNA expression studies following treatment of the HNSCC cell line FaDu with LBH589 reveal down-regulation of genes required for chromosome congression and segregation (SMC2L1), sister chromatid cohesion (DDX11), and kinetochore structure (CENP-A, CENP-F, and CENP-M); these LBH589-induced changes in gene expression coupled with the down-regulation of MYC and BIRC5 (survivin) provide a plausible explanation for the early mitotic arrest and cell death observed. When LBH589-induced changes in gene expression were compared with gene expression profiles of 41 primary HNSCC samples, many of the genes that were down-regulated by LBH589 showed increased expression in primary HNSCC, suggesting that some patients with HNSCC may respond to treatment with LBH589.

Invasion of tumor cells into the local stroma is an important component in cancer progression. Here we report studies of the in vivo invasion of head and neck squamous cell carcinoma (HNSCC) cells in response to applied gradients of a growth factor [epidermal growth factor (EGF)] and a chemokine (CXCL12), using orthotopic floor-of-mouth models. Analysis of the invading cells indicated that >75% of them were tumor cells, about 15% macrophages, and

Bortezomib-induced quiescence of multiple myeloma cells is associated with changes in the expression of p21CIP/KIP and the H3K27-methyltransferase EZH2. Most multiple myeloma (MM) patients respond positively to the proteasome inhibitor (PI) bortezomib (Velcade). However, there are high rates of relapse after variable remission periods. It is still unclear whether these periods of remission are due to residual cells that after bortezomib treatment enter dormancy or simply due to a small residual population that slowly proliferate until patients becoming symptomatic. It is therefore important to determine whether minimal residual disease (MRD) can be rendered quiescent by bortezomib. We had shown that in MM cells, bortezomib induced ER-stress and quiescence. However, the mechanism for the induction and maintenance of this quiescent state is unclear. We hypothesized that the prolonged quiescence might involve prototypical cell cycle inhibitors and that epigenetic mechanisms sustain the growth arrest. Quantification of viable cells using immunofluorescence microscopy showed a 6-fold increase in p21 protein after a 24-hour pulse treatment with bortezomib followed by washout. p21 and p27 were also persistently induced 2-fold and 1.5-fold respectively along with the downregulation of CDK6 3 days after the drug washout. This suggests that quiescence onset is triggered by cell cycle inhibitor induction that block the G1 to S transition. This correlated with the previously reported reduction in Rb phosphorylation in bortezomib-surviving MM cells. When we tested the levels of the histone H3 methyl-transferase EZH2 and H3K27me3, these were strongly downregulated in the quiescent MM cells. EZH2 depletion was previously linked to p21-mediated senescence in melanoma. Our results suggest that changes in histone methylation and cell cycle inhibitor induction may be a mechanism regulating bortezomib-induced quiescence. Further studies will validate whether these mechanisms are active in patients by analyzing clinical samples from MM patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5680. doi:1538-7445.AM2012-5680

The vast majority of cancer related deaths are due to metastasis. However, our understanding of this process is limited. This is clearer when we consider that these lesions can develop years or decades after successful primary tumor treatment. These long periods of clinical remission can be explained by minimal residual disease (i.e. Disseminated Tumor Cells, DTCs) entering a non-productive or dormant state. However, our limited knowledge of these events has curtailed the development of strategies to prevent metastasis. Key questions in this field are: How does early dissemination start and what are the mechanisms? How does the tumor microenvironment aid this process? Are primary tumor (PT) niches responsible for DTCs growth or quiescence? What role does the microenvironment in the metastatic niche play in determining the timing of DTC dormancy? In patients, DTCs that are non-proliferative can be found in sites where they usually form secondary lesions or in sites where they never do. Thus despite being able to disseminate these DTCs are “growth-suppressed” by certain target organ microenvironments. Several mechanisms are proposed to explain clinical dormancy. The absence of proliferation markers in DTCs in patients and experimental models suggest that solitary DTCs are quiescent. However, angiogenic dormancy or immune system-mediated tumor mass dormancy might also be responsible for maintaining residual disease dormant. At least three potential scenarios might explain DTC dormancy. 1) DTCs from invasive cancers activate stress signals in response to the dissemination process and/or a growth suppressive target organ microenvironment, inducing dormancy. 2) Therapy and/or microenvironmental stress conditions acting on PT cells carrying specific gene signatures primes newly DTCs to enter dormancy. Thus, specific PT “stress microenvironments” might trigger long-term DTC dormancy. 3) Lesions pathologically defined as non-invasive carry cells able to undergo micro-invasion and disseminate. Here although these DTCs were able to disseminate and survive they are unfit for expansion in secondary sites. Perhaps occasional cell divisions allow them to progress via epigenetic and genetic pathways to a fully metastatic cell able to grow in secondary sites. We propose that DTC dormancy is ultimately a survival strategy that when blocked will prevent metastasis. Below we expand on the first two scenarios. For additional details on the third scenario please see. DTC dormancy and the target organ microenvironment. Solitary DTCs in target organs establish interactions with the ECM, immune cells and the vascular system. This and the pattern of metastasis proposed by the seed and soil theory suggests that microenvironments in the target organ can determine metastatic growth vs. dormancy. Our work shows that in squamous carcinoma cells (HEp3) reduced urokinase (uPA) receptor (uPAR) expression deactivated α5β1-integrins and this made these cells incapable of binding efficiently to fibronectin. This resulted in reduced Src-FAK and EGFR signaling but also in p38α/β activation. This caused these tumor cells to enter a state of dormancy characterized by a prolonged G0-G1 arrest. Others have reproduced these findings showing that loss of β1-integrin or FAK signaling in breast cancer models can also induce dormancy and that Src-MLKC signaling can prevent dormancy. In addition, an enriched collagen-I lung microenvironment can trigger intravenously delivered tumor cells to exit from dormancy as solitary cells. On the other hand environments rich in fibrillar collagen-I can induce quiescence of melanoma cells via activation of the discoidin domain receptor 2 and p15INK4b induction. These results imply that stress signaling induced either by therapies or by a restrictive (i.e. fibrotic or non-fibrotic target tissues depending on the tumor type) tissue microenvironments could activate dormancy (or its interruption) in DTCs. Our work in the HEp3 model also revealed that activation of p38α/β, while inhibiting ERK1/2 signaling activates an unfolded protein response (UPR). These signals induce survival and quiescence of dormant HEp3 (D-HEp3) cells. D-HEp3 cells in vivo enter a deep G0-G1 arrest due to p21 and p27 upregulation. At least 3 transcription factors (TFs), p53, BHLHB3/41/Sharp1 and NR2F1 were induced by p38α/β and required for dormancy in vivo. Bone marrow derived dormant HEp3 cells displayed a low ERK/p38 signaling ratio and induction of these TFs. Interestingly, metastasis suppressor genes (MSGs) like MKK4 and MKK6 activate p38, BHLHB3 is a target of p38 and Nm23-H1 (another MSG) appears to function via the downregulation of EDG2 LPA receptor signaling through ERK1/2. Thus, different mechanisms converge on the regulation of the ERK/p38 signaling ratio to dictate DTC fate. Overall these studies provide clues as to potential mechanisms to induce or maintain dormancy or eradicate DTCs by targeting their survival signals. Primary tumor microenvironments as determinants of DTC fate. Gene signatures identified in PTs predict long-term metastatic relapse more than a decade later and in the absence of the PT carrying the signature. Gene profiles from the tumor stroma also predict patient outcome. This suggests that a crosstalk between PT cells and their microenvironment in primary sites can dictate distant disease progression. One interpretation is that genes signatures in the PT and the microenvironment determine DTC fate. Since symptomatic metastasis show homogeneous progression, the gene signatures in the PT might provide information on how those individual or groups of genes influence dormancy of DTCs. Modeling how the genes influence DTC survival and quiescence or subsequent angiogenesis or interaction with the immune system might reveal how they regulate minimal residual disease. Importantly, determining whether signatures derived from circulating tumor cells (CTCs) (i.e. recently intravasated tumor cells) are more or equally informative than the PT signatures might justify characterizing CTCs vs. DTCs (i.e. CTCs that already lodged and reside in target organs). Another potential example of the primary tumor influencing DTC behavior proposed that CTCs might return to the PT in a self-seeding process and this helps “breed” more aggressive variants that colonize target organs. These studies showed that aggressive variants of MDA-MB-231 breast cancer cells were highly efficient in disseminating and cross-seeding contra lateral tumors. The less aggressive variants of different cancer cell lines were less efficient in the seeding self/cross-seeding process. These data suggest that development of a more aggressive metastatic progeny requires the ability of PTs to attract their own CTCs back and that these tumor cells can efficiently re-colonize the PT. It is unclear how this happens after PT surgery, but it is possible that these events take place when multiple metastases co-exist. It will be interesting to determine whether CTCs from patients with early and advanced lesions carry already these “self-seeding” signatures. An additional scenario of a PT microenvironment influencing DTC behavior is that of systemic tumor instigation. This model proposes that PTs can influence the growth of otherwise-indolent DTCs or micrometastases. This is though to occur by mobilizing bone-marrow cells into the stroma of the indolent lesions. These results suggest that growth and proliferation of poorly aggressive tumors (dormant DTCs and/or micrometastasisβ) can be regulated on a systemic level by endocrine factors released by certain instigating tumors. However, it remains unclear how metastasis are instigated years or decades after patients underwent PT surgery. Regardless of their origin we propose that characterization of DTCs would be the most relevant because they carry the aggregate information of their origin, how treatment influenced their adaptation and/or selection and ultimately how the target organ dictated their adaptation and/or selection. The challenges presented by the problem of cancer dormancy are significant and studying DTCs and dormant disease is difficult. But the benefits of unraveling the inherent complexity of this step of metastasis biology should be of great impact for cancer patients. Grant Support: Samuel Waxman Cancer Research Foundation Tumor Dormancy Program, NIH/National Cancer Institute (CA109182, CA163131), NIEHS (ES017146) and NYSTEM grant to J.A.A-G. M.S.S. is supported by a DoD-BCRP Grant-10904826. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY17-02. doi:1538-7445.AM2012-SY17-02

We have further examined the hypoxia-induced gene expression in the sickle transgenic mouse model with high expression of human βS and α globin that is homozygous for the mouse β-major deletion, NY1DD (Fabry et al, PNAS89:12159, 1992) exposed to hypoxia (8% O2/92% N2) for 2 or 4 days vs normoxic conditions. Gene expression in the kidney was studied using cDNA expression microarrays, analyzed by GenePix software and the Significance Analysis of Microarrays (SAM) program, and validated by real-time PCR and immunocytochemistry. Data analyzed by hierarchical clustering (that segregates genes expressed in the hypoxic kidneys vs the normoxic kidney) and SOTA (self-organizing tree algorithm) revealed the effect of hypoxia on different clusters of genes. In one cluster, the effect of hypoxia on heme oxygenase-1 (HO-1), was particularly striking. HO-1 is slightly downregulated in normoxic kidneys and becomes up-regulated 2 and 4 fold on days 2 and 4 of hypoxia. HO-1 was also observed upon immunocytochemical staining with HO-1 antibodies (Calbiochem) in kidney sections from 4 kidneys harvested from 2 mice exposed to either 2 or 4 days hypoxia (4 mice total) and 2 kidneys each from a C57BL and a NY1DD mouse harvested under room air. Mice sacrificed under room air had negligible staining for HO-1. However, NY1DD mice maintained under hypoxia for 2 days had moderate HO-1 staining in some of the proximal tubules near the surface of the cortex and sickled cells in the vasculature. NY1DD mice exposed to 4 days of hypoxia had more intense staining for HO-1 in the proximal tubules that extend deeper towards the medulla. Histological examination of kidney sections confirmed red cell congestion and sickling predominantly in medullary vessels where hypoxia is maximal. HO-1, the rate-limiting enzyme in the heme degradation pathway, is inducible in the kidney (Pimstone et al, J Clin Invest50:2042, 1971) and increased in sickle mouse kidneys (Nath et al, Am J Path158:893, 2001). HO-1 also protects cells from oxidant damage. Other very up-regulated genes include myosin Vb (involved in membrane remodeling) and lipocalin 2 (present in distal tubules/collecting ducts and modulates the cellular response to injury). Genes that were down-regulated include calbindin 28K (a cytosolic vitamin D dependent calcium binding protein that regulates calcium homeostasis potentially disrupted by hypoxia), metallothionein 2 (a zinc binding protein and oxidant scavenger that blocks the most reactive oxidant species including peroxynitrite and superoxide radical), fructose bisphosphatase (a gluconeogenic enzyme located in the proximal nephron and a marker for glomerular damage) and the sodium/calcium exchanger (downregulated in renal ischemia/reperfusion injury as a mechanism to protect the cell from unregulated calcium influx). Identification of genetic modifiers of renal pathophysiology in sickle cell anemia is the first step in understanding the clinical heterogeneity among SS patients. These studies may also reveal novel metabolic pathways in the SS kidney and may, therefore, hold potential for therapeutic intervention.

Background The mechanisms allowing residual multiple myeloma (MM) cells to persist after bortezomib (Bz) treatment remain unclear. We hypothesized that studying the biology of bortezomib-surviving cells may reveal markers to identify these cells and survival signals to target and kill residual MM cells. Methods We used H2B-GFP label retention, biochemical tools and in vitro and in vivo experiments to characterize growth arrest and the unfolded protein responses in quiescent Bz-surviving cells. We also tested the effect of a demethylating agent, 5-Azacytidine, on Bz-induced quiescence and whether inhibiting the chaperone GRP78/BiP (henceforth GRP78) with a specific toxin induced apoptosis in Bz-surviving cells. Finally, we used MM patient samples to test whether GRP78 levels might associate with disease progression. Statistical analysis employed t-test and Mann-Whitney tests at a 95% confidence. Results We report that Bz-surviving MM cells in vitro and in vivo enter quiescence characterized by p21CIP1 upregulation. Bz-surviving MM cells also downregulated CDK6, Ki67 and P-Rb. H2B-GFP label retention showed that Bz-surviving MM cells are either slow-cycling or deeply quiescent. The Bz-induced quiescence was stabilized by low dose (500nM) of 5-azacytidine (Aza) pre-treatment, which also potentiated the initial Bz-induced apoptosis. We also found that expression of GRP78, an unfolded protein response (UPR) survival factor, persisted in MM quiescent cells. Importantly, GRP78 downregulation using a specific SubAB bacterial toxin killed Bz-surviving MM cells. Finally, quantification of Grp78high/CD138+ MM cells from patients suggested that high levels correlated with progressive disease. Conclusions We conclude that Bz-surviving MM cells display a GRP78HIGH/p21HIGH/CDK6LOW/P-RbLOW profile, and these markers may identify quiescent MM cells capable of fueling recurrences. We further conclude that Aza + Bz treatment of MM may represent a novel strategy to delay recurrences by enhancing Bz-induced apoptosis and quiescence stability. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1460-1) contains supplementary material, which is available to authorized users.