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Supplemental Figure 5. RB status is retained in biopsy proven local recurrence.

Purpose: Perturbations in the retinoblastoma pathway are over-represented in advanced prostate cancer; retinoblastoma loss promotes bypass of first-line hormone therapy. Conversely, preliminary studies suggested that retinoblastoma-deficient tumors may become sensitized to a subset of DNA-damaging agents. Here, the molecular and in vivo consequence of retinoblastoma status was analyzed in models of clinical relevance.Experimental Design: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAPC4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of retinoblastoma using shRNA). Multiple mechanisms were interrogated including cell cycle, apoptosis, and DNA damage repair. Transcriptome analysis was performed, validated, and mechanisms discerned. Cell survival was measured using clonogenic cell survival assay and in vivo analysis was performed in nude mice with human derived tumor xenografts.Results: Loss of retinoblastoma enhanced the radioresponsiveness of both hormone-sensitive and castrate-resistant prostate cancer. Hypersensitivity to ionizing radiation was not mediated by cell cycle or p53. Retinoblastoma loss led to alteration in DNA damage repair and activation of the NF-κB pathway and subsequent cellular apoptosis through PLK3. In vivo xenografts of retinoblastoma-deficient tumors exhibited diminished tumor mass, lower PSA kinetics, and decreased tumor growth after treatment with ionizing radiation (P < 0.05).Conclusions: Loss of retinoblastoma confers increased radiosensitivity in prostate cancer. This hypersensitization was mediated by alterations in apoptotic signaling. Combined, these not only provide insight into the molecular consequence of retinoblastoma loss, but also credential retinoblastoma status as a putative biomarker for predicting response to radiotherapy. Clin Cancer Res; 20(21); 5468–82. ©2014 AACR.

Supplemental Figure 2. RB deficiency promotes enhanced apoptotic pathway in response to ionizing radiation

Supplemental Figure 1. RB status does not impinge on the alterations of cell cycle after radiation

Supplemental Figure 4. PLK3 expression promotes apoptotic cell death in the setting of RB loss.

Supplemental Figure 3. NFκB is retained in the cytoplasm in the presence of an IκB dominant negative.

IMPORTANCE Dry eye disease (DED) is common, but little is known about factors contributing to symptoms of dry eye, given the poor correlation between these symptoms and objective signs at the ocular surface.OBJECTIVE To explore whether pain sensitivity plays a role in patients' experience of DED symptoms.DESIGN, SETTING, AND PARTICIPANTS A population-based cross-sectional study of 1635 female twin volunteers, aged 20 to 83 years, from the TwinsUK adult registry.MAIN OUTCOMES AND MEASURES Dry eye disease was diagnosed if participants had at least 1 of the following: (1) a diagnosis of DED by a clinician, (2) the prescription of artificial tears, and/or (3) symptoms of dry eyes for at least 3 months. A subset of 689 women completed the Ocular Surface Disease Index (OSDI) questionnaire. Quantitative sensory testing using heat stimulus on the forearm was used to assess pain sensitivity (heat pain threshold [HPT]) and pain tolerance (heat pain suprathreshold [HPST]).RESULTS Of the 1622 participants included, 438 (27.0%) were categorized as having DED. Women with DED showed a significantly lower HPT (P=.03) and HPST (P=.003)-and hence had higher pain sensitivity-than those without DED. A strong significant association between the presence of pain symptoms on the OSDI and the HPT and HPST was found (P=.008 for the HPT and P=.003 for the HPST). In addition, participants with an HPT below the median had DED pain symptoms almost twice as often as those with an HPT above the median (31.2% vs 20.5%; odds ratio, 1.76; 95% CI, 1.15-2.71; P=.01).CONCLUSIONS AND RELEVANCE High pain sensitivity and low pain tolerance are associated with symptoms of DED, adding to previous associations of the severity of tear insufficiency, cell damage, and psychological factors. Management of DED symptoms is complex, and physicians need to consider the holistic picture, rather than simply treating ocular signs.