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Supplementary Table from Inhibition of LSD1 Attenuates Oral Cancer Development and Promotes Therapeutic Efficacy of Immune Checkpoint Blockade and YAP/TAZ Inhibition

Supplementary Table 1. RNAi used in the study.

Supplementary Figure S3. Gene set enrichment analysis of the YAP/TAZ-activated genes.

Supplementary Figure from Inhibition of LSD1 Attenuates Oral Cancer Development and Promotes Therapeutic Efficacy of Immune Checkpoint Blockade and YAP/TAZ Inhibition

Supplementary Table 2. Antibodies used in the study.

Lysine-specific demethylase 1 (LSD1) is a histone demethylase that contributes to the etiology of oral squamous cell carcinoma (OSCC) in part by promoting cancer stem cell phenotypes. The molecular signals regulated by LSD1, or acting with LSD1, are poorly understood, particularly in the development of OSSC. In this study, we show that conditional deletion of the Lsd1 gene or pharmacologic inhibition of LSD1 in the tongue epithelium leads to reduced development of OSCC following exposure to the tobacco carcinogen 4NQO. LSD1 inhibition attenuated proliferation and clonogenic survival and showed an additive effect when combined with the YAP inhibitor Verteporfin. Interestingly, LSD1 inhibition upregulated the expression of PD-L1, leading to immune checkpoint inhibitor therapy responses.Implications:Collectively, our studies reveal a critical role for LSD1 in OSCC development and identification of tumor growth targeting strategies that can be combined with LSD1 inhibition for improved therapeutic application.

Supplementary Table 5. Gene list from the cluster analysis in Fig 5B.

Supplementary Table 3. qPCR primers used in the study.

Supplementary Table 4. Microarray data from siCTL, siTAZ, siYAP, and siYAP/TAZ-treated SCC2 cells.

Supplementary Table 6. Hyper-enrichment analysis of YAP/TAZ-regulated molecular signature.

Supplementary Figure 1 from Overexpression of DPAGT1 Leads to Aberrant N-Glycosylation of E-Cadherin and Cellular Discohesion in Oral Cancer

Supplementary Figure 2 from Overexpression of DPAGT1 Leads to Aberrant N-Glycosylation of E-Cadherin and Cellular Discohesion in Oral Cancer

Supplementary Figure Legends 1-2 from Overexpression of DPAGT1 Leads to Aberrant N-Glycosylation of E-Cadherin and Cellular Discohesion in Oral Cancer

Lysine-specific demethylase 1 (LSD1) is a histone demethylase that contributes to the etiology of oral squamous cell carcinoma (OSCC) in part by promoting cancer stem cell phenotypes. The molecular signals regulated by LSD1, or acting with LSD1, are poorly understood, particularly in the development of OSSC. In this study, we show that conditional deletion of the Lsd1 gene or pharmacologic inhibition of LSD1 in the tongue epithelium leads to reduced development of OSCC following exposure to the tobacco carcinogen 4NQO. LSD1 inhibition attenuated proliferation and clonogenic survival and showed an additive effect when combined with the YAP inhibitor Verteporfin. Interestingly, LSD1 inhibition upregulated the expression of PD-L1, leading to immune checkpoint inhibitor therapy responses. Implications: Collectively, our studies reveal a critical role for LSD1 in OSCC development and identification of tumor growth targeting strategies that can be combined with LSD1 inhibition for improved therapeutic application.

Extracellular lysyl oxidases (LOX and LOXL1–LOXL4) are critical for collagen biosynthesis. LOXL2 is a marker of poor survival in oral squamous cell cancer. We investigated mechanisms by which tumor cell secreted LOXL2 targets proximal mesenchymal cells to enhance tumor growth and metastasis. This study identified the first molecular mechanism for LOXL2 in the promotion of cancer via its enzymatic modification of a non-collagenous substrate in the context of paracrine signaling between tumor cells and resident fibroblasts. The role and mechanism of active LOXL2 in promoting oral cancer was evaluated and employed a novel LOXL2 small molecule inhibitor, PSX-S1C, administered to immunodeficient, and syngeneic immunocompetent orthotopic oral cancer mouse models. Tumor growth, histopathology, and metastases were monitored. In vitro mechanistic studies with conditioned tumor cell medium treatment of normal human oral fibroblasts were carried out in the presence and absence of the LOXL2 inhibitor to identify signaling mechanisms promoted by LOXL2 activity. Inhibition of LOXL2 attenuated cancer growth and lymph node metastases in the orthotopic tongue mouse models. Immunohistochemistry data indicated that LOXL2 expression in and around tumors was decreased in mice treated with the inhibitor. Inhibition of LOXL2 activity by administration of PXS-S1C to mice reduced tumor cell proliferation, accompanied by changes in morphology and in the expression of epithelial to mesenchymal transition markers. In vitro studies identified PDGFRβ as a direct substrate for LOXL2, and indicated that LOXL2 and PDGF-AB together secreted by tumor cells optimally activated PDGFRβ in fibroblasts to promote proliferation and the tendency toward fibrosis via ERK activation, but not AKT. Optimal fibroblast proliferation in vitro required LOXL2 activity, while tumor cell proliferation did not. Thus, tumor cell-derived LOXL2 in the microenvironment directly targets neighboring resident cells to promote a permissive local niche, in addition to its known role in collagen maturation.

Oral squamous cell carcinoma (OSCC) is a prevalent form of cancer that develops from the epithelium of the oral cavity. OSCC is on the rise worldwide, and death rates associated with the disease are particularly high. Despite progress in understanding the mutational and expression landscape associated with OSCC, advances in deciphering these alterations for the development of therapeutic strategies have been limited. Further insight into the molecular cues that contribute to OSCC is therefore required. Here, we show that the transcriptional regulators YAP ( YAP1 ) and TAZ ( WWTR1 ), which are key effectors of the Hippo pathway, drive protumorigenic signals in OSCC. Regions of premalignant oral tissues exhibit aberrant nuclear YAP accumulation, suggesting that dysregulated YAP activity contributes to the onset of OSCC. Supporting this premise, we determined that nuclear YAP and TAZ activity drives OSCC cell proliferation, survival, and migration in vitro , and is required for OSCC tumor growth and metastasis in vivo . Global gene expression profiles associated with YAP and TAZ knockdown revealed changes in the control of gene expression implicated in protumorigenic signaling, including those required for cell cycle progression and survival. Notably, the transcriptional signature regulated by YAP and TAZ significantly correlates with gene expression changes occurring in human OSCCs identified by The Cancer Genome Atlas (TCGA), emphasizing a central role for YAP and TAZ in OSCC biology. Implications: This study defines a YAP/TAZ-regulated transcriptional program in OSCC and reveals novel roles for nuclear YAP/TAZ activity in the onset and progression of this cancer. Mol Cancer Res; 13(6); 957–68. ©2015 AACR . This article is featured in Highlights of This Issue, [p. 955][1] [1]: /lookup/volpage/13/955?iss=6

Objective We report intraoral granulomatous foreign body reactions in patients treated with calcium hydroxylapatite (CHA) or poly- l -lactic acid (PLA). Study Design Clinical and histopathologic data were obtained from 25 patients who developed orofacial nodules or swelling after dermal filler injections. Results All 25 patients were women aged 35 to78 years (median, 55 years). All had a history of injection of CHA (n = 13) or PLA (n = 12) to the lips, nasolabial area, or mental area. Two patients developed cutaneous nodules at the sites of injections; all others presented with intraoral nodules (labial/buccal or vestibular mucosa) distant from the site of injections, suggestive of filler migration. Five of 21 cases presented with pain. Histopathologically, CHA presented as a diffuse mass of mauve-gray or beige, nonrefractile spherules, and PLA as rice- or spindle-shaped, geometric, refractile bodies within circumscribed nodules. Conclusions Cutaneous injections of CHA and PLA fillers may induce granulomatous reactions presenting as intraoral nodules distant from the injection sites.

Phosphaturic mesenchymal tumor (PMT) is a rare neoplasm that has been associated with oncogenic osteomalacia. This tumor secretes fibroblast growth factor-23 (FGF-23). Although other mesenchymal tumors can cause oncogenic osteomalacia, PMT is the most common mesenchymal neoplasm associated with oncogenic osteomalacia and accounts for 80% of such cases. Most patients are adults and the tumor can affect both men and women. The most common location for the tumor is the lower extremities followed by the head and neck area. Patients typically present with diffuse bone pain, bone fractures, and progressive muscle weakness. The laboratory studies usually reveal increased levels of FGF-23, hyperphosphaturia, increased alkaline phosphatase, normal serum calcium and parathyroid hormone levels, normal to low levels of 1,25-dihydroxyvitamin D and hypophosphatemia. The four common phosphaturic mesenchymal tumor microscopic subtypes are: phosphaturic mesenchymal tumor mixed connective tissue variant (PMTMCT), osteoblastoma-like variant, ossifying fibroma-like variant, and non-ossifying fibroma-like variant. The small size of this slow growing tumor and the non-specific clinical presentation may present a diagnostic challenge to clinicians. We present a case of a 48-year-old Haitian male who was diagnosed with PMT in the right angle of the mandible. Fewer than 15 cases of PMT presenting in the oral cavity have been reported in the literature. The clinical presentation, the laboratory findings, imaging characteristics, and the histopathologic features for this case are discussed along with the molecular genetic aspects, treatment and prognosis for this rare neoplasm.

Objectives Benign alveolar ridge keratosis (BARK) is a benign hyperkeratosis that occurs as a poorly demarcated white papule or plaque on the retromolar area or edentulous alveolar ridge mucosa caused by trauma. Histopathologic features are identical to cutaneous lichen simplex chronicus, a condition that results from chronic habitual skin scratching/picking. P53 protein is a tumor suppressor protein that plays a critical role in DNA repair. P53 protein has been shown to be present within 5-25% of the basal cell nuclei in normal oral mucosa and reactive lesions. The objective of this study is to report on the histopathologic features of BARK and to explore P53 expression pattern. Study Design Cases of BARK were identified from the biopsy service of the Harvard School of Dental Medicine from January, 2016 to December, 2017. Randomly selected cases were studied for the presence of P53. Results There were 174 cases comprising 119 males and 55 females (2.2:1; M:F) with a median age of 57 years (range 15-86). The majority were in the sixth (31.0%) and seventh (29.3%) decades. There were 112 (64.4%) cases on the retromolar pad and 62 (35.6%) on the edentulous alveolar mucosa; 27 (15.5%) cases were bilateral. Histopatho-logically, the oral mucosa showed hyperkeratosis often with wedge-shaped hypergranulosis and occasional focal parakeratosis. The epithelium exhibited mild to moderate acanthosis and slight surface undulations or papillomatosis, with tapered rete ridges, often confluent at the tips. The study for P53 performed in 11 cases showed less than 25 % nuclear positivity. Conclusion BARK is a distinct benign clinicopathologic entity caused by friction that is the intraoral counterpart of cutaneous lichen simplex chronicus with which it shares similar histopathologic features. It is not a mere hyperkeratosis which would relegate it to the clinical entity of leukoplakia, and which is a potentially malignant condition.

The perineurioma is an infrequently encountered benign peripheral nerve sheath tumor composed of a clonal proliferation of perineurial cells. Rare cases of perineurioma have been reported in the oral cavity. An extraneural sclerosing perineurioma arising in the buccal mucosa of a 17-year-old male is presented. Histopathologically, the tumor is composed of a well circumscribed nodular proliferation of spindle cells arranged in a storiform growth pattern, in some areas subtly arranged around vascular channels. The tumor cells reveal positive immunostaining for epithelial membrane antigen (EMA), collagen type IV and vimentin, and negative immunostaining for S-100 protein, consistent with a perineurial origin. To the best of our knowledge, this case represents the first report of an extraneural sclerosing perineurioma involving the oral cavity.

Objective Resorbable collagen membranes (RCMs) are commonly used by oral surgeons, periodontists, and endodontists for multiple purposes. We report 6 cases of RCMs that did not resorb as expected and describe the histopathologic features. Study Design Cases of an unusual fibrillar foreign material were noted in biopsy specimens curetted from bone. Hematoxylin-eosin and Masson trichrome stains were performed. Clinicians were contacted for detailed clinical information. Results There were 3 men and 3 women. RCMs presented as hyalinized, paucicellular, delicate eosinophilic fibrils or a meshwork without a foreign body reaction. They were refractile and stained for Masson trichrome as expected. These RCMs persisted longer than expected (2-6 weeks) in 3 cases and may have retarded healing in 5 cases. Conclusions Although RCM is supposed to be fairly rapidly resorbable, this material sometimes persists within wound sites without any obvious foreign body reaction and may retard healing.